Nikos C. Apostolopoulos - Stretch Intensity and The Inflammatory Response - A Paradigm Shift-Springer International Publishing (2018)

Nikos C.
Apostolopoulos
Stretch Intensity
and the
Inflammatory
Response: A
Paradigm Shift
Stretch Intensity and the Inflammatory Response:
A Paradigm Shift
Nikos C. Apostolopoulos
Stretch Intensity
and the Inflammatory
Response: A Paradigm Shift
Nikos C. Apostolopoulos
University of Toronto
Toronto, ON, Canada
ISBN 978-3-319-96799-8 ISBN 978-3-319-96800-1 (eBook)

https://doi.org/10.1007/978-3-319-96800-1
Library of Congress Control Number: 2018961999
© Springer Nature Switzerland AG 2018

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I dedicate this book to my constant
companions, my daughters, Thalia Sofia
and Justine Francis.
Preface
“The true nature of knowledge is experiment…

What is now prove’d was once only imagin’d”
William Blake 1757–1827
Although nature may have buried her secrets deep, she has not entirely hidden them.
As scientists, we are curious, often investigating for information and insight in order
to enlighten ourselves with that which lies beyond her veil. In order to gain a
glimpse, we use imagination, intuition, and invention, but mostly enthusiasm and
perseverance, the qualities of an undying desire to make clear that which has so
often eluded us, but through intuition, we know truly exists. At times, we walk and
occupy the nether zone between reality and illusion in order to bridge the gap
between the outer and the inner worlds. This is a sensitive chaos that we attempt to
explain through the language of mathematics (numbers, equations, statistics, etc.),
believing that this will make the invisible visible. The belief is that, by solving the
formation of this now visible pattern, frozen in time and space through observation,
one will obtain a better idea into nature’s function and activity. Similar to Parzifal’s
quest for the Holy Grail and how he refused to be raised in the dark forest, we chal-
lenge ourselves to learn and discover the mysteries and the inner workings of the
human body. Parzifal was faced with the dilemma of whether to respond to the
plight of the fisher king, Astofar, or to hold steadfastly true to the code of the knights.
His adherence to the code cost him several more years of wondering before fulfill-
ing his task. Just seeing the Holy Grail is not enough; we have to ask about it, mak-
ing inquiries while approaching our task with an open and unbiased mind, breaking
old thought patterns and codes in order to advance, to be the biographers of a much-
needed paradigm shift.
Toronto, ON, Canada Nikos C. Apostolopoulos
vii
Acknowledgements
“Truth is the offspring of silence and meditation. I keep the subject constantly before me
and wait ‘til the first drawings open slowly, by little and little, into a full and clear light.”
– Sir Isaac Newton
While researching and writing this manuscript, I found myself meandering in and
out of numerous moments of contemplation, for such was the nature of the work at
hand. Bathed in silence, I had pensive periods that allowed for necessary reflections
as to how best to proceed. During these moments, my thoughts drifted to and about
those who had gone before, the pioneers who paved the way. Enquiring about the
property of things, the forming of hypotheses, and the designing of experiments,
they were responsible for making clear that which was observed or intuitively
believed. With enthusiasm, perseverance, and the need to know, they were instru-
mental in laying the foundation from which this manuscript was made manifest.
The quote by Sir Isaac Newton best sums up their influence: “If I have seen further
it is only by standing on the shoulders of giants…”. Although the opportunity has
yet to present itself to meet with these individuals, I would nonetheless like to
acknowledge their contributions to my thought processes and to science as a whole.
Distinguished Professors Donald E. Ingber (Harvard University) and James
G. Tidball (UCLA) have had a profound influence on my knowledge and under-
standing of the response of the cell and muscle tissue. Professor Ingber’s compel-
ling work on the concept of mechanotransduction has been pivotal in formulating
my thoughts about the effect of mechanical force (i.e., stretching intensity) on mus-
cle tissue and cells. In other words, how the magnitude of the stretch can manifest a
biochemical response. Professor James G. Tidball provided the stepping stone from
which to relate the effects of this mechanical stress on the recovery and regeneration
of muscle tissue. For the inflammatory response, both acute and chronic, the cyto-
kines, and C-reactive proteins, I am indebted to the work of Professor Emeritus
Irving Kushner (Case Western University). His work has made these complex con-
cepts simple and coherent. The groundbreaking research on calpains by Professor
Angelo Belcastro (York University, Canada) was the keystone, connecting my
hypothesis with the magnitude of stretching intensity to inflammation. Moving
ix
x Acknowledgements
across the pond to Europe, two distinguished researchers stand out: Clinical
Professor Michael Kjaer (Institute of Sports Medicine, Copenhagen) and Professor
Dr. Matthias Chiquet (University of Bern). Their research has been immensely valu-
able in formulating thoughts regarding the response of the extracellular matrix
(ECM) to mechanical stress. The ECM provides the structural framework, contrib-
uting to the mechanical stability of tissues as well as acting as a substrate for cell
adhesion. When a mechanical stimulus is imparted on the body, this imparts infor-
mation to the cells, resulting in their subsequent response. However, a common
denominator to a lot of the material mentioned above is the work of the pioneer and
visionary, the late Buckminster Fuller. Science owes a major debt to this individual.
His abridgement of the concepts of tension and integrity, tensegrity, is the founda-
tion for the response of the cell to mechanical force(s). Continuous tensional behav-
iour, an interplay between tension and compression from the macro to the cellular
level, is the basis for how the magnitude of stretching affects muscle tissue and
cells, and is responsible for the significance of the inflammatory response.
In conclusion, no acknowledgement would be complete without mentioning
those who have had a direct impact on me. Besides my immediate family and clos-
est friends, a heartfelt gratitude goes out to Professor Emeritus Dr. Jack Taunton
(University of British Columbia, Canada) and Professor Michael J. Plyley (Brock
University, Canada). Both were instrumental in pushing me out of my comfort zone,
encouraging me to test the waters in order to grow as a scientist and therapist. To my
coach and mentor, Andy Higgins (University of Toronto), our numerous discussions
and his advice held me steadfast and true to my goals both professionally and per-
sonally. Finally, like everything, true friendship possesses a quality of its own. It is
unique, it listens openly, it is not afraid to speak the truth, and it is not envious but
selfless, with a great willingness to be truly present. These characteristics define my
oldest friend and esteemed colleague, Professor Peter Pericles Trifonas (University
of Toronto). During times of great trepidation, our numerous walks and talks were
the light at the end of the tunnel, and for this I am truly indebted.
Contents
1 Introduction�� 1
References�� 3
2 Literature Review�� 5
Introduction�� 5
The Definition of Stretching and Its Measurement�� 6
Classification of Stretches�� 9
Static�� 9
Ballistic�� 10
Dynamic�� 11
Proprioceptive Neuromuscular Facilitation (PNF)�� 11
Macroscopic and Microscopic Information Processing Levels:
Macroscopic—Muscle, Tendon, and Myotendon Unit (MTU) �� 12
Muscles, Tendons, and MTU �� 14
Macroscopic and Microscopic Information Processing
Levels: Microscopic—Force, Cells, Tissue, and the Extracellular
Matrix (ECM)�� 42
Force, Cells, and Tissue �� 42
The Extracellular Matrix (ECM) �� 44
Conclusion �� 50
The Strain Factors of Stretching: Intensity, Duration,
and Frequency�� 51
Inflammation�� 53
Neutrophils, Macrophages, Cytokines, and Acute
Phase Response�� 55
Inflammation and Exercise�� 59
Mechanotransduction�� 87
Stretching and Inflammatory Cells�� 92
References�� 94
xi
xii Contents
3 Study One: Acute Inflammatory Response to Stretching�� 131

Methods�� 132
Participants�� 132
Power Calculations�� 133
Procedures�� 133
Biomarkers�� 135
Statistical Analysis �� 135
Results�� 136
RMANOVA�� 136
Effect Sizes�� 136
Discussion�� 139
References�� 140
4 Study Two: Stretch Intensity vs. Inflammation:
Is There a Dose-Dependent Association? �� 145
Methods�� 146
Power Calculations�� 146
Blood Biomarkers�� 149
Results�� 150
RMANOVA�� 150
Effect Sizes�� 151
Secondary Analysis: Linear Regression (30–90 and 60–90%
Maximum ROM)�� 151
5 The Effect of Different Passive Static Stretching Intensities
on Perceived Muscle Soreness and Muscle Function
Recovery Following Unaccustomed Eccentric Exercise:
A Randomised Controlled Trial�� 159
Methods�� 161
Results�� 166
Contents xiii
Perceived Muscle Soreness�� 166

Magnitude-Based Inference Approach�� 166
Eccentric Peak Torque�� 168
Isometric Peak Torque �� 174
Permission�� 178
6 Summary Discussion�� 183
Acute Inflammatory Response to Stretching �� 183
Stretch Intensity Vs. Inflammation: Is there a Dose-Dependent
Association?�� 185
The Effects of Different Passive Static Stretching Intensities
on Perceived Muscle Soreness and Muscle Function Recovery
Following Unaccustomed Eccentric Exercise: A Randomised Trial �� 186
7 Limitations�� 189
Limitations of Studies (Table 7.1)�� 189
Blood Collection�� 189
Methodology�� 191
Compliance�� 192
Nutrition�� 192
Mental Stress�� 193
Age�� 193
Summary�� 193
8 Future Research �� 197
Further Research�� 198
Further Research Based on Studies�� 198
Ultrasonography�� 199
Reactive Oxygen Species�� 200
Musculoskeletal Disorders and Pain�� 201
Recovery and Regeneration �� 202
Relaxation Response�� 203
9 Conclusion�� 209
xiv Contents
Section One: Forms�� 211
Section Two: Samples of Raw Data for Studies�� 221
Glossary�� 225
Index�� 227
List of Figures
Fig. 2.1 Classification of stretching�� 9

Fig. 2.2 The sarcomere�� 16
Fig. 2.3 The sarcomere depicting the four filaments (in red)�� 17
Fig. 2.4 Titin relationship to Z-disc and M-line �� 22
Fig. 2.5 Z-disc proteins partially located in Z-disc (associations
and function) �� 26
Fig. 2.6 Z-disc proteins fully located in Z-disc (associations and
function)�� 27
Fig. 2.7 Z-disc proteins, intra- and extramyofibril �� 27
Fig. 2.8 Intermediate filaments and costameres �� 29
Fig. 2.9 Costamere associations �� 32
Fig. 2.10 Myotendon unit�� 39
Fig. 2.11 Forces�� 43
Fig. 2.12 Classification of skeletal muscle ECM �� 45
Fig. 2.13 Key players in mechanical homeostasis�� 48
Fig. 2.14 Summary of cytokines�� 57
Fig. 2.15 Acute phase response�� 58
Fig. 2.16 Relationship of IL-6 relative to concentric and eccentric
non-damaging exercise �� 61
Fig. 2.17 Comparison of muscle damage (a) vs. habitual exercise
(b) induced cytokines (note: during muscle damage IL-6
release is preceded by TNF-α)�� 62
Fig. 2.18 Cytokine cascade related to muscle damage�� 66
Fig. 2.19 The four stages with regard to muscle-damaging exercise
and inflammatory response �� 66
Fig. 2.20 Stage1: Muscle damage�� 67
Fig. 2.21 Stage 2: Autolytic calpain �� 67
Fig. 2.22 Stage 3: Adhesion and transendothelial migration of leukocytes �� 67
Fig. 2.23 Stage 4: Inflammatory cells�� 68
Fig. 2.24 Stage 4: Inflammatory cells—neutrophils�� 68
xv
xvi List of Figures
Fig. 2.25 Stage 4: Inflammatory cells—pro-inflammatory macrophages

(phagocytic)�� 68
Fig. 2.26 Stage 4: Inflammatory cells—anti-inflammatory macrophages
(non-phagocytic) �� 69
Fig. 2.27 Measurement of histological changes�� 71
Fig. 2.28 Subsequent steps associated with the autolysis by calpain�� 74
Fig. 2.29 Transendothelial migration �� 76
Fig. 2.30 Expression of anti-inflammatory cytokines�� 86
Fig. 2.31 Hierarchy of systems (macro to micro)�� 89
Fig. 3.1 Methodology schematic of intervention(s) �� 134
Fig. 3.2 Change of hsCRP over time (h)�� 137
Fig. 4.1 Knee immobiliser�� 147
Fig. 4.2 Experimental set-up�� 147
Fig. 4.4 Linear regression between 30 and 90% maximum ROM
and hsCRP post �� 152
and hsCRP post �� 153
and hsCRP 24 h post �� 153
and hsCRP 24 h post �� 154
Fig. 5.2 Passive static stretching exercises �� 164
Fig. 5.3 Perceived muscle soreness (raw)�� 165
Fig. 5.4 Distribution of perceived muscle soreness as a function of time�� 166
Fig. 5.5 Eccentric peak torque (raw)�� 173
Fig. 5.6 Histogram of eccentric peak torque difference �� 173
Fig. 5.7 Eccentric peak torque 72 vs. 0 h (scatter plot)�� 174
Fig. 5.8 Isometric peak torque (raw). *LIS low-intensity passive static
stretching, HIS high-intensity passive static stretching�� 174
Fig. 5.9 Histogram of isometric peak torque difference�� 175
Fig. 5.10 Isometric peak torque 72 vs. 0 h (scatter plot)�� 175
List of Tables
Table 2.1 Time scale of inflammatory cell response in relation

to the phases of muscle regeneration post muscle damage�� 69
Table 2.2 Sequence of inflammatory cells �� 82
Table 3.1 Mean (SD) of raw and LN transformed blood biomarkers
in relation to time and intervention(s)�� 137
Table 3.2 Descriptive statistics and effect sizes for all between
conditions and within conditions comparisons�� 138
Table 4.1 Mean (SD) measurement and effect sizes for hsCRP�� 150
Table 5.1 Measurements of perceived muscle soreness, eccentric
and isometric peak torque over 72 h post-unaccustomed
eccentric exercise protocol (values are mean ± SD; n = 10 per
group) �� 168
Table 5.2 Changes in perceived muscle soreness, eccentric
and isometric peak torque in the low-intensity passive static
stretching (LIS) vs. high-intensity passive static stretching
(HIS) group (n = 10 per group)�� 169
and isometric peak torque in the low-intensity passive static
stretching (LIS) vs. control (CON) group (n = 10 per group)�� 171
and isometric peak torque in the high-intensity passive static
stretching (HIS) vs. control (CON) group (n = 10 per group) �� 172
Table 6.1 Summary of studies�� 184
Table 7.1 Summary of studies and scheduled time periods for blood
collection�� 190
xvii
xviii List of Tables
Table A.1 Age, mass, height, and hsCRP values�� 221

Table A.2 Age, mass, height, and hsCRP values for 30%, 60%,
and 90% maximum ROM�� 222
Table A.3 Perceived muscle soreness and eccentric and isometric peak
torque�� 222
Chapter 1
Introduction
Nothing happens in the animate world without a high degree of cooperation of the
individual parts of a biological system, a close relationship between function and
the underlying structure. Biological systems are energetically open, being in a posi-
tion to exchange with their surroundings both energy and material, but more impor-
tantly, information. By means of a synergistic process, systems respond to a
mechanical insult on the body and “upconvert” the energy created at the molecular
level (biochemical response) to macroscopic energy forms (i.e. muscle contraction,
stretching, and various associated patterns) suggesting the importance of and the
influence of the environment (Haken, 2004). One complex system in the body and
the focus of this manuscript is inflammation and the inflammatory process, a mes-
senger pathway, a biochemical information circuit, notifying the body of potential
challenges which have disturbed its homeostasis. It is a coordinated response aimed
at maintaining or restoring tissue integrity (Rubartelli, Lotze, Latz, & Manfredi,
2013). Disruption of this communication network creates an instability point where
the system changes its spatio-temporal pattern or function in reaction to a variety of
conditions including the presence of pathogens, as well as chemical, thermal, and
mechanical stressors (Butterfield, Best, & Merrick, 2006). For instance, acute mus-
cle damage, caused by crush injuries, overloading, and/or strain injuries initiates a
sequence of cellular responses defining inflammation (Tidball, 1995), with the pres-
ence of necrotic and apoptotic cells further triggering an inflammatory response
(Rock & Kono, 2008).
The activation of this process in response to trauma, injury, and other insults
stimulates the recruitment of blood leukocytes, the activation of tissue macrophages,
and a production of a host of other mediators (Ward & Lentsch, 1999). Although the
focus in the present manuscript is on the effect of a physical insult (i.e. magnitude
of stretching) and the incitement of inflammation, this activation is not limited to
just a physical perturbation. Mental and emotional stress can provoke neuroendo-
crine and brain neurotransmitter changes interpreted by the brain as being stressors,
triggering an inflammatory response, contributing to the development of depression
(Anisman & Merali, 2003).
© Springer Nature Switzerland AG 2018 1

N. C. Apostolopoulos, Stretch Intensity and the Inflammatory Response:
A Paradigm Shift, https://doi.org/10.1007/978-3-319-96800-1_1
2 1 Introduction
Inflammation can be either acute or chronic, but in reality it represents a

continuum (Young, Stewart, & O’Dowd, 2010), dependent upon the intensity and/
or duration of the stimulus responsible for its initiation. The acute inflammatory
response—through the acute phase response induced in the liver—refers to a gener-
alised acknowledgement of the body to tissue injury (i.e. chemical, thermal, or
mechanical), with the sole purpose of healing (Smith, 1991). The major character-
istic of this process is the mobilisation of inflammatory cells to the affected site,
with the resolution being the elimination and subsequent return of these inflamma-
tory cells to homeostasis (Maskrey, Megson, Whitfield, & Rossi, 2011; Serhan
et al., 2007). In contrast, chronic inflammation is characterised by a persistent
inflammatory stimuli (Serhan et al., 2007) orchestrated by numerous chemical
mediators derived from the injured and damaged tissue (Young et al., 2010). The
continued tenacity of the stimuli without any interruption, its elimination, or its total
clearance results in the development of a vicious circle observed in the pathophysi-
ology of many human diseases, including cancer, chronic viral infections, autoim-
munity, as well as acquired and inborn genetic disorders (Rubartelli et al., 2013).
However, unlike chronic inflammation, acute inflammation is self-limiting and does
resolve, with the return of tissue homeostasis and integrity, allowing the individual
to return to a somewhat normal function. The current work is primarily focused on
the acute response.
Stretching has been defined as an external and/or internal force with the intent of
increasing muscle flexibility and/or joint ROM (Maffulli, King, & Helms, 1994;
Weerapong, Hume, & Kolt, 2004). The sensing of this force or any force by the cells
of the musculoskeletal tissue is fundamental for their proper function. Analyses of
physical aspects within the environment responsible for influencing the response of
the cell to the environment are force and geometry. With regard to geometry, sub-
strate elasticity or stiffness changes to the cell are influenced in response to the
elasticity or the stiffness of the tissue, the change of physical shape. These two
aspects are responsible for facilitating the biochemical response of the cells to an
external mechanical signal (i.e. stretching).
The stretching parameters consist of intensity, duration, frequency, and stretch-
ing position (Apostolopoulos, Metsios, Flouris, Koutedakis, & Wyon, 2015;
Marschall, 1999). Unlike duration and frequency, which are quantifiable (i.e. how
long to hold and how often to repeat the stretch), intensity is qualitative explaining
why it has attracted little scientific interest thus far. Regardless of this qualitative
nature, the magnitude of the physical force(s) (i.e. intensity) applied to the muscle
during stretching is a catalyst for tissue damage and inflammation as observed in
animal studies (Pizza, Koh, Mcgregor, & Brooks, 2002).
Pain and/or discomfort present during the act of stretching may imply tissue
damage especially when considering the definition of pain as suggested by the
International Association for the Study of Pain. According to this organisation, pain
is “an unpleasant sensory and emotional experience associated with actual or poten-
tial tissue damage, or described in terms of such damage” (Merskey & Bogduk,
1994).
References 3
An injured muscle typically undergoes stages of degeneration, inflammation,

regeneration, and fibrosis, depending, partly, on the severity of the injury (Li,
Cummins, & Huard, 2001; Urso, 2013). According to Nordenstrom, locally injured
tissue liberates catabolic energy giving rise to a physicochemical potential differ-
ence relative to the surrounding non-injured tissue (Nordenstrom, 1983). This injury
is responsible for tissue polarisation, a physiochemical gradient, which the organ-
ism tries to equalise spontaneously in order to decrease the entropy in the system
caused by the rupture and subsequent death of the myofibres. This results in the
infiltration of inflammatory cells to the extracellular matrix (ECM) and the sur-
rounding vasculature (Urso, 2013).
The ECM is a functionally diverse entity consisting of rigid, elastic, as well as
wet components imparting informational signals to the cells enabling them to adjust
their physiology accordingly (Mecham, 2001). It is not monosyllabic, but a com-
plex and intricate arrangement, dictated by the interrelationship of its component
parts (collagens, elastin and microfibrillar proteins, adhesive glycoproteins, and
proteoglycans) and the cells it interacts with (Mecham, 2001). Activation of the
acute inflammatory response is characteristic of this intricate interrelationship
designed to re-establish tissue integrity and homeostasis. This is orchestrated by
numerous chemical mediators derived from the injured tissue(s), with the most
important indicated at their site(s) of action (Wheater & Burkitt, 1996). Reference
will be made to such mediators, specifically to the acute phase protein, C-reactive
protein (CRP), and the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α. The
current manuscript will examine how the magnitude and rate of force developed
during stretching may moderate and modulate inflammation and the inflammatory
response, with reference made to these blood biomarkers. Much of the research on
inflammation has focused on inhibiting it with drugs. Though this approach has
merit, since the degree and extent of the inflammatory response may result in further
injury to muscle, preventing it may hinder recovery (Ebbeling & Clarkson, 1989;
Mackey et al., 2007). Therefore, the purpose of this project was to investigate the
importance of and the role that stretching intensity may play regarding the recovery
of athletes from training, competition, and injury, as well as in the rehabilitation of
individuals suffering from various musculoskeletal disorders (i.e. rheumatoid arthri-
tis (RA), osteoarthritis (OA), etc.).
References
Anisman, H., & Merali, Z. (2003). Cytokines, stress and depressive illness: Brain-immune interac-
tions. Annals of Medicine, 35, 2–11.
Apostolopoulos, N., Metsios, G. S., Flouris, A. D., Koutedakis, Y., & Wyon, M. (2015). The rel-
evance of stretch intensity and position - a systematic review. Frontiers in Psychology, 6, 1128.
Butterfield, T. A., Best, T. M., & Merrick, M. A. (2006). The dual roles of neutrophils and mac-
rophages in inflammation: A critical balance between tissue damage and repair. Journal of
Athletic Training, 41, 457–465.
4 1 Introduction
Ebbeling, C. B., & Clarkson, P. M. (1989). Exercise-induced muscle damage and adaptation.
Sports Medicine, 7, 207–234.
Haken, H. (2004). Synergetics: Introduction and advanced topics. Berlin, Germany: Springer.
Li, Y., Cummins, J., & Huard, J. (2001). Muscle injury and repair. Current Opinion in
Orthopaedics, 12, 409–415.
Mackey, A. L., Kjaer, M., Dandanell, S., Mikkelsen, K. H., Holm, L., Dossing, S., et al. (2007).
The influence of anti-inflammatory medication on exercise-induced myogenic precursor cell
responses in humans. Journal of Applied Physiology, 1034, 425–431.
Maffulli, N., King, J., & Helms, P. (1994). Training in elite young athletes (the training of young
athletes (TOYA) study): Injuries, flexibility and isometric strength. British Journal of Sports
Medicine, 28, 123–136.
Marschall, F. (1999). Wie beinflussen unterschiedliche dehnintensitaten kurzfristig die verander-
ung der bewegungsreichweite? (Effects of different stretch-intensity on the acute change of
range of motion). Deutsche Zeitschrift fur Sportmedizin, 50, 5–9.
Maskrey, B. H., Megson, I. L., Whitfield, P. D., & Rossi, A. G. (2011). Mechanisms of resolu-
tion of inflammation - a focus on cardiovascular disease. Arteriosclerosis, Thrombosis, and
Vascular Biology, 31, 1001–1006.
Mecham, R. P. (2001). Overview of extracellular matrix. Current Protocols in Cell Biology,
00.10.1, 10.1.1–10.1.14.
Merskey, H., & Bogduk, N. (1994). Classification of chronic pain. Seattle, WA: IASP Press.
Nordenstrom, B. E. W. (1983). Biologically closed electric circuits-clinical, experimental and
theoretical evidence for an additional circulatory system. Stockholm, Sweden: Nordic Medical
Publications.
Pizza, F. X., Koh, T. J., Mcgregor, S. J., & Brooks, S. V. (2002). Muscle inflammatory cells after
passive stretches, isometric contractions, and lengthening contractions. Journal of Applied
Physiology, 92, 1873–1878.
Rock, K. L., & Kono, H. (2008). The inflammatory response to cell death. Annual Review of
Pathology, 3, 99–126.
Rubartelli, A., Lotze, M. T., Latz, E., & Manfredi, A. (2013). Mechanisms of sterile inflammation.
Frontiers in Immunology, 2013, 1–2.
Serhan, C. N., Brain, S. D., Buckley, C. D., Gilroy, D. W., Haslett, C., O’Neill, L. A., et al. (2007).
Resolution of inflammation: State of the art, definitions and terms. FASEB, 21, 325–332.
Smith, L. L. (1991). Acute inflammation: The underlying mechanism in delayed onset muscle
sorenes? Medicine and Science in Sports and Exercise, 23, 542–551.
Tidball, J. G. (1995). Inflammatory cell response to acute muscle injury. Medicine and Science in
Sports and Exercise, 27, 1022–1032.
Urso, M. L. (2013). Role of inflammation in skeletal muscle, connective tissue, and exertional
injuries: To block or not to block? Anti-inflammatory interventions and skeletal muscle injury:
Benefit or detriment? Journal of Applied Physiology, 115, 920–928.
Ward, P. A., & Lentsch, A. B. (1999). The acute inflammatory response and its regulation. Archives
of Surgery, 134, 666–669.
Weerapong, P., Hume, P., & Kolt, G. (2004). Stretching: Mechanisms and benefits for sport perfor-
mance and injury prevention. The Physical Therapy Review, 9, 189–206.
Wheater, P. R., & Burkitt, H. G. (1996). Wheater's basic histopathology: A colour atlas and text.
New York: Chruchill Livingstone.
Young, B., Stewart, W., & O’Dowd, G. (2010). Wheater's basic pathology: A text, atlas and review
of histopathology. New York: Churchill Livingstone.
Chapter 2
Literature Review
Introduction
The human body is an “open” system exchanging matter, energy, and information
with its immediate environment. This exchange often results in the manifestation of
internal processes and transformations, defined by such concepts as homeostasis,
self-regulation, allostasis, and autopoieses. These processes allude to the body’s
innate ability to monitor its mechanical usage and its subsequent response through
use of several mechanisms, based on the exchange of information between the
external and internal environments. According to Hannan and Freeman (Hannan &
Freeman, 1977), homeostasis is based on information exchanged between the sys-
tem and the external environment allowing the system to maintain a state of equilib-
rium over time. Self-regulation is an adaptive mechanism by which the system
maintains a balanced condition within its structural limits and through the exchange
of information with its external environment (Beer, 1975), while allostasis refer-
ences a transformational concept, the idea that maintenance of stability occurs
through change (Mcewen & Wingfield, 2003). It suggests that through this process
the organism actively adjusts to both predictable and unpredictable events (Mcewen
& Wingfield, 2003). With autopoieses, the predominant feature is the self-
organisation of a system’s ability to stimulate a selective mechanism designed to
align its internal complexity with the complex external environment (Maturana &
Varela, 1975). Although each of these transformational processes appear to be dif-
ferent in defining the relationship(s) between the internal and external environ-
ments, a common thread is the exchange and processing of information, the
formative response of the body to either a physical or a mental stress. Within this
current work, the focus is on the exchange of information between a physical pro-
cess (i.e. stretching), specifically the body’s response(s) to the magnitude, and rate
of such a stimulus. This information exchange alludes to the concept of mechano-
transduction, “the process of converting physical forces into biochemical signals

6 2 Literature Review
and integrating these signals into (a) cellular response” (Huang, Kamm, & Lee,
2004). More will be discussed later.
The practice of stretching is widespread in the arts (i.e. dancing), the general fit-
ness population (i.e. stretching programmes), as well as sports (Taylor, Dalton,
Seaber, & Garrett, 1990). Stretching is viewed as a physical strategy aimed at
improving flexibility (Weerapong, Hume, & Kolt, 2004), performance (Handel,
Horstmann, Dickhuth, & Gulch, 1997; Wilson, Elliot, & Wood, 1992), pain relief,
and recovery from injury (Chandler et al., 1990; Jackson et al., 1978; Maffulli,
King, & Helms, 1994). In addition, it has become the universal strategy for injury
prevention in sports (Thacker, Gilchrist, Stroup, & Kimsey, 2004). Athletes, coaches,
trainers, and medical professionals (physicians, physiotherapists, etc.) often recom-
mend stretching to enhance performance and prevent injury and thus regard it as an
essential component of numerous physical training programmes (Caplan, Rogers,
Parr, & Hayes, 2009) and rehabilitation protocols (Feland, Myrer, & Merrill, 2001;
Malliaropoulos, Papalexandris, Papalada, & Papacostas, 2004). A review by
Prentice has indicated that four stretching methods are commonly used for sport
activities—static, ballistic, dynamic, and proprioceptive neuromuscular facilitation
(PNF) (Prentice, 1985).
The Definition of Stretching and Its Measurement
Stretching, a movement applied by an external and/or internal force stressing con-

nective and muscle tissue mechanically, is used to increase muscle flexibility and/or
joint ROM (Martins et al., 2013; Weerapong et al., 2004). It depends on the active
and passive tension of the muscle, the MTU, and the proprioceptors of the muscu-
loskeletal system, the muscle spindles, and the Golgi tendon organs (GTO) (Abdel-
Aziem, Draz, Mosaad, & Abdelraou, 2013; Guissard & Duchateau, 2006; Knudson,
2006; Nikolaou, Macdonald, Glisson, Seaber, & Garrett, 1987). In addition, non-
muscle elements such as ligaments, tendons, and bones provide physiological con-
straints for maximal muscle stretch in skeletal muscle (Agarkova, Ehler, Lange,
Schoenuer, & Perriard, 2003). Active tension is the interaction of the actin and myo-
sin filaments of the muscle, with passive being the elongation of the connective
tissue beyond its resting length (Knudson, 2006). Both of these define the length-
dependent properties of the muscle, which is strongly related to stretching, for the
interaction of each implies that exercise interventions, like stretching, may have a
complex effect on skeletal muscle, dependent on the interaction of the tissues and
the nature of the training stimulus (Knudson, 2006). In other words, when muscle is
stretched by various stretching techniques, this may account for changes in the
active and passive tension of the muscle (Apostolopoulos, Metsios, Flouris, &
Koutedakis, 2015). An understanding of the molecular pathways governing the abil-
ity of the muscle to respond to a functional demand presents a better comprehension
of the influence of stretch intensity on the musculoskeletal tissue(s), since the cells
are influenced by physical force(s).
The Definition of Stretching and its Measurement 7
The force generated during stretching is a load, with load referring to the
magnitude and the rate of force developed (Elliot, 2006). An understanding of how
force affects soft and connective tissue at both the macroscopic (muscle, tendons,
and myotendon unit) and microscopic (sarcomere, myofibrillar proteins, ECM)
level provides a better insight as to how the magnitude and rate of force generated
during stretching affects the body as a whole. This knowledge is pivotal in design-
ing a proper stretching protocol aimed at increasing athletic performance or reha-
bilitation from musculoskeletal pain and injury.
At the macroscopic level, change due to stretching is primarily functional, while
at the microscopic level, it is reflected in the cells and the ECM of the muscles, the
tendons, and the MTU. A reduction in reflex sensitivity of the skeletal muscle with
repeated stretching alters its mechanical tension resulting in a functional change
(Avela, Kyrolainen, & Komi, 1999; Proske, Morgan, & Gregory, 1993). With ten-
dons, stretching may change their dynamic behaviour, confirmed in findings from
animal studies demonstrating that the elasticity of tendons is changeable through
stretching (Viidik, 1969; Witvrouw, Mahieu, Roosen, & Mcnair, 2007). Regardless
of the functional or structural transformation associated with stretching, an under-
standing of how the body adapts to this force, both macroscopically and micro-
scopically, is essential and will be discussed further.
The cytoskeletal organisation of the cells, in other words, the contractile forces
of the microfilaments and the compression resistance of microtubules, is responsi-
ble for the cell response to any static and physical force sensed. This response is
defined by the varying levels of resistance influenced by the cell’s attachment and
adherence to the ECM and its relationship to adjacent cells. In addition, the cell
itself also generates force influencing both its function and shape. This highlights
the influence directed at the responsiveness of individual cells to any external
force(s) (i.e. fluid flow, stretching), as well as the cellular responses to cell exerted
forces connected to and influenced by the extracellular microenvironment (Discher,
Janmey, & Wang, 2005).
By referring to both the macroscopic and microscopic levels, in particular the
integration and transference of information through the numerous levels from the
external environment to the cytoskeleton of the cell, an understanding is achieved
regarding the degree of force (intensity) developed during stretching and how this
may cause a structural and mechanical change to the tissue. The sensing of force by
cells is critical for their proper function, being fundamental in mediating a load
transfer, as well as sustaining an interaction between the cells and tissue critical for
the interface of function and homeostasis (Benjamin, Evans, & Copp, 1986; Lu &
Jiang, 2006; Woo et al., 1988). Movement from the macro to the micro represents a
continuum, an intracellular transfer of information explaining the body’s ability to
adapt to the external environment. This information is particularly important if one
considers that the magnitude of force generated during stretching may determine
the effectiveness of stretching regarding the structural adjustment of the connective
and muscle tissue to this externally applied force(s). An effectiveness may help in
resolving trauma to the connective and muscle tissue or create and exacerbate the
damage to these structures.
The increase in ROM about a joint with stretching is facilitated by the synchronised
interaction(s) of the many types of soft tissue (muscle, tendons, etc.) and their rela-
tion to bone (joints) (Shellock & Prentice, 1985). In addition to this physiological
relationship, a functional/behavioural one exists, the placement of the body in a
proper position facilitating a more effective stretch. This allows for a better isolation
of a muscle group (i.e. hamstring, quadriceps, etc.) and their particular joint(s) (i.e.
knee, ankle, hips, etc.), ensuring the ability to control and apply the appropriate
amount of a mechanical stress (i.e. stretching) aimed at increasing the ROM while
limiting tissue damage (Abdel-aziem et al., 2013; Apostolopoulos et al., 2015). In
other words, the position assumed during stretching may influence the magnitude of
the force generated prior to and during the stretch potentially altering the response
of the muscle and tendon tissue (Abdel-aziem et al., 2013; Apostolopoulos et al.,
2015).
Range of motion is an important factor in human athletic performance and in
many traumatic or orthopaedic conditions (Maffulli et al., 1994). The degree and
distribution of ROM in individuals may be essential in maximising physical perfor-
mance as well as the genesis and presentation of injuries (Jackson et al., 1978). It
can be specific to each side of the body even in the same joint (Chandler et al., 1990;
Maffulli et al., 1994).
Flexibility is typically measured in degrees of full ROM to the point of mild
discomfort from a starting position (Luttgens & Hamilton, 1997). Goniometers and
inclinometers have been used to measure it, respectively. The former is widely used
in clinical settings measuring any limitations while documenting the effectiveness
of the intervention (Gajdosik & Bohannon, 1987). The two-arm goniometer is most
commonly used for the evaluation of ROM (Lea & Gerhardt, 1995). Its usefulness,
however, has been questioned since the starting position, the long axis of the limb,
the centre of rotation, and the vertical and horizontal positions can only be visually
estimated (Nussbaumer et al., 2010). Nevertheless, its reliability is high, confirming
its continued use in clinical settings (Mayerson & Milano, 1984; Rothstein, Miller,
& Roettger, 1983).
Similar to the goniometer, an inclinometer is portable and lightweight requiring
little training to assess joint ROM (Kolber, Fuller, Marshall, Wright, & Hanney,
2012). Two types of inclinometers exist: gravity-based and digital. The former
makes use of a liquid compartment or magnet establishing zero degrees when posi-
tioned in the vertical. With constant gravity as a reference point, this inclinometer
can be used to assess joint mobility and ROM (Dejong, Nieuwboer, &
Aufdemkampe, 2007; Kolber, Vega, Widmayer, & Cheng, 2011). Digital inclinom-
eters are costly, with a major disadvantage being the requirement of the clinician or
examiner to establish the zero point accurately in order to minimise the risk of
measurement error prior to use (Kolber et al., 2012). Overall, the inclinometer also
possesses good reliability when strict measurement protocols are adhered too
(Kolber et al., 2011, 2012).
Classification of Stretches 9
Classification of Stretches
Stretching is classified as static, dynamic, and pre-contraction (Fig. 2.1). Within

these categories there are active (self-stretch) and passive (with or without a partner)
for static stretching, active and ballistic for dynamic stretching, and PNF for pre-
contraction stretching (Page, 2012; Weerapong et al., 2004). These categories are
not universal with some authors suggesting the existence of only four types of
stretches: static, ballistic, dynamic, and PNF (Behm & Chaouachi, 2011; Shellock
& Prentice, 1985). Regardless of the classification, the common denominator for
each technique is the placement of a certain load(s) on the muscles and the joint(s)
being targeted.
Static
Static stretching is commonly employed to improve joint mobility and prevent con-
tracture (Nakamura, Ikezoe, Takeno, & Ichihashi, 2012). It usually involves moving
the limb to the end of its ROM and holding this position for 15–60 s at a point of
discomfort or pain (Bandy, Irion, & Briggler, 1997; Behm & Chaouachi, 2011;
Feland, Myrer, & Merrill, 2001; Sady, Wortman, & Blanke, 1982; Smith, 1994). It
is performed passively with use of a partner or therapist or, actively, with the indi-
vidual self-stretching. The primary use of static stretching has been for increasing
the ROM about a joint (Bandy et al., 1997; Feland, Myrer, & Merrill, 2001), attrib-
uted to changes in the length and stiffness of the MTU of the limb being stretched
(Behm & Chaouachi, 2011; Power, Behm, Farrel, Carroll, & Young, 2004). However,
Fig. 2.1 Classification of stretching. (Published with kind permission of copyright © Nikos
C. Apostolopoulos 2018. All rights reserved)
a controversy exists whether this change is due to a decrease in MTU stiffness

(Wilson, Wood, & Elliot, 1991) or is it primarily an increase in the tolerance to
stretching (Law et al., 2009; Magnusson, Simonsen, Aagaard, Sorensen, & Kjaer,
1996).
Static stretching takes advantage of the inverse myotatic reflex promoting muscle
relaxation, thereby causing a further stretch and an increase in ROM (Feland, Myrer,
Schulthies, Fellingham, & Measom, 2001). Other benefits have been the reduction
of pain and injury (Safran, Seaber, & Garrett, 1989; Smith, 1994), a decrease in
soreness (High, Howley, & Franks, 1989), as well as improved performance (Young
& Behm, 2002). However, systematic reviews looking at the impact of static stretch-
ing for prevention of sports injury risk concluded that static stretching does not
reduce injury rates (Bo Lauersen, Bertelsen, & Bo Andersen, 2014; Small,
Mcnaughton, & Matthews, 2008) or change the muscle-tendon mechanical proper-
ties (Freitas et al., 2017). In addition, static stretching may affect the development
of explosive force, jumping, as well as sprint performance (Winchester, Nelson,
Landin, Young, & Schexnayder, 2008; Young & Behm, 2003). Nevertheless, a study
has observed that static stretching prior to competition(s) does not negatively impact
the vertical jump performance in trained women (Unick, Kieffer, Cheesman, &
Feeney, 2005). These controversies, whether static stretching improves performance
or not and reduces pain and soreness or whether the increase in ROM is due to
stretch tolerance or a change in MTU stiffness, reflect the lack of our current under-
standing and thus the need for further research.
Ballistic
Similar to static stretching, with “ballistic” stretching, the joint is passively moved
to its maximum ROM, whereupon a dynamic “ballistic” movement is applied on the
stretched structure at the end of the ROM (Konrad & Tilp, 2014). This is in the form
of a bouncing rhythmic motion using the momentum of a swinging body segment to
lengthen the muscle (Bandy et al., 1997; Mahieu et al., 2007), which activates the
stretch reflex resulting in a contraction of the muscle under stretch. This rapid
change in tension, increasing with the magnitude and rate of the stretch, may pro-
duce a strain or rupture of the tissue making this form of stretching disadvantageous
for improving ROM (ACSM, 2006; Page, 2012; Shrier & Gossal, 2000). It is note-
worthy that articles referring to ballistic stretching as being disadvantageous con-
sisted of a clinical commentary (Page, 2012), a nonsystematic literature review
(Shrier & Gossal, 2000), and guidelines as suggested by the American College of
Sports Medicine (ACSM, 2006). The views expressed in these articles represent
findings associated with the notion that the rapid alternating “bouncing” at the end
ROM increases risk of injury, although relevant good quality studies are currently
lacking. In contrast, articles supporting ballistic stretching are all randomised clini-
cal trials concluding that ballistic stretching is beneficial for increasing hamstring
flexibility (Kumar & Chakrabarty, 2010; Morcelli, Oliveira, & Navega, 2013) or in
Classification of Stretches 11
combination with other techniques presents a more appropriate approach with

regard to training and rehabilitation (Mahieu et al., 2007). Similar to static stretch-
ing, the aforementioned discrepancy illustrates the need for more robust research to
provide better and relevant information and create a better understanding of the
various stretching techniques.
Dynamic
Dynamic stretching involves moving a limb through its full ROM to the end ranges
for several times (Page, 2012). This method has been introduced as a better alterna-
tive to static stretching for increasing ROM (Murphy, 1994). With dynamic stretch-
ing, movement begins from the neutral position of the joint, performed slowly and
deliberately (Bandy et al., 1997). Murphy suggests that if the limbs are moved too
quickly, a tendency to swing the limb exists which may cause the stretch reflex to be
elicited at the endpoint of the movement during the lengthening of the muscle. The
slow deliberate movement of the limb back to the neutral position is executed with
use of an eccentric contraction of the muscle. This contraction by the antagonist
muscle results in the relaxation of the lengthening muscle due to the principle of
reciprocal inhibition. In other words, the muscle is reflexively inhibited (Murphy,
1994).
Proprioceptive Neuromuscular Facilitation (PNF)
This stretching technique developed to improve muscle elasticity to treat neurologi-

cal dysfunctions (Knott & Voss, 1968) has been shown to have a positive effect on
both active and passive ROMs (Hindle, Whitcomb, Briggs, & Hong, 2012).
Currently two techniques, collectively referred to as PNF procedures (Etnyre &
Lee, 1987) are used, the contract-relax and the contract-relax-antagonist-contract
(Condon & Hutton, 1987; Hindle et al., 2012; Page, 2012), with each using a voli-
tional contraction in order to increase ROM. The rationale behind the contract-relax
technique is that the successive maximal excitations of the motor neurons during the
volitional contractions will reflexively promote their subsequent inhibition (Kabat,
1950). With contract-relax-antagonist-contract, contraction and relaxation of the
muscle to be stretched (i.e. the stretching of the target muscle) is followed by a
concentric contraction of the opposing muscle. Activation of the opposing muscle is
believed to reduce activation of the target muscle through reciprocal innervation
(Hindle et al., 2012; Kabat, 1950; Knott & Voss, 1968; Voss, 1967). An example of
a paired target and opposing muscle is the quadriceps and hamstring muscles,
respectively. Although, it has been reported that PNF results in a greater increase in
ROM compared to static stretching (Magnusson, Simonsen, Aagaard, et al., 1996;
Magnusson, Simonsen, Dyhre-Poulsen, et al., 1996; Sady et al., 1982), with the
effects lasting 90 min or more (Funk, Swank, Mikla, Fagan, & Farr, 2003), the
mechanism(s) thought to facilitate this increase in muscle length has been ques-
tioned (Chalmers, 2004; Shrier & Gossal, 2000). In general, the literature suggests
that activation of the opposing muscle motoneurones results in the simultaneous
excitation of the Ia-inhibitory interneurons which synapse the target muscle moto-
neurones, resulting in a decrease in the neural activity of the target muscle (Hultborn,
Illert, & Santini, 1976; Sharman, Cresswell, & Riek, 2006). The inhibition of the
proprioceptive structures in the target muscle (i.e. Golgi tendon organ), in response
to the contraction, or shortening of the opposing muscle, is responsible for the
lengthening of the target muscle fibres (Hindle et al., 2012; Sharman et al., 2006).
However, instead of observing a decrease in electromyography activity as suggested
by reciprocal inhibition, inferring a reduction in active force production which
would resist the lengthening of the muscle (Chalmers, 2004), the muscle exhibits
both an active electromyography and a subsequent increase in muscle stiffness
(Moore & Hutton, 1980; Shrier & Gossal, 2000). Therefore, increases in ROM of
the target muscle (i.e. quadriceps) occurred despite the opposing muscle (i.e. ham-
string) being under considerable tension suggesting that the PNF techniques
(contract-relax and contract-relax-antagonist-contract) failed to evoke sufficient
relaxation in the muscle to overcome tension generated by the stretch (Osternig,
Robertson, Troxel, & Hansen, 1987). Regardless of this controversy, PNF proce-
dures have consistently been observed to enhance ROM compared to either static or
ballistic stretching (Etnyre & Abraham, 1986; Feland, Myrer, Schulthies, et al.,
2001; Funk et al., 2003; O’hara, Cartwright, Wade, Hough, & Shum, 2011; Sady
et al., 1982; Wallin, Ekblom, Grahn, & Nordenborg, 1985).
acroscopic and Microscopic Information Processing Levels:

M
Macroscopic—Muscle, Tendon, and Myotendon Unit (MTU)
Introduction
Since the time of the French philosopher and mathematician Rene Descartes (1596–
1650) and the British natural philosopher Sir Isaac Newton (1643–1727), biologists
have adopted the approach of studying the mechanism(s) of the body in response to
the environment from both an ontological and epistemological level (Ahn, Tewari,
Poon, & Phillips, 2006; Mazzochi, 2008). The former refers to the concept of the
organism and how it is made with the latter focused on understanding those things
that define it. To accomplish this, scientists embraced the concepts of simplification
and reductionism, suggesting that the complexity of the body can be resolved by
both an analysis and a reduction of everything to its simplest components. In other
words, biological systems can be explained largely by referring to the physical and
chemical properties of their individual components (Westerhoff & Palsson, 2004).
Macroscopic and Microscopic Information Processing Levels: Macroscopic—Muscle… 13
In vitro studies have been instrumental in discovering, observing, and determining

individual components (cells), providing merit to the reductionist approach, albeit
that the procedures are conducted in a controlled environment outside of the living
organism. They were important in elucidating the assembly of myofibrils, a dynamic
interaction dependent on the coordinated integration of myosin, actin, associated
proteins, and other cytoskeletal elements. However, we need to be cognisant that
these protein interactions frequently include an unmanageable number of binary
interactions with uncertain functional significance (Ayalon et al., 2011). The whole
organism is a dynamic, adaptive, and interactive network, a study of the relations
between components possessing the ability for the development of new collective
properties in order to maintain allostasis and stability through change (Mcewen &
Wingfield, 2003).
An understanding of complex systems is accessible through in vivo studies.
Unlike in vitro, they refer to an experimentation using a whole living organism as
opposed to a partial or dead organism. They enable the observation of the various
degrees of interactions of components at multiple levels, disclosing the behaviour of
the system as a whole. The exchange between all levels provides the system with the
plasticity to rapidly adapt to the changing environmental situations (Coffey, 1998),
suggesting that the whole is far from equilibrium. It is an interplay between order
and randomness absorbing as well as dissipating energy in the quest to maintain
itself (Biebricher, Nicolis, & Schuster, 1995). For instance, compared to the study
of individual muscle fibres through in vitro, a muscle in vivo is subjected to substan-
tial transverse and bending loads that would not be present in isolation (Burkholder,
2007). These loads are imposed through pressures in contact with and attachment to
bone, tendons, fascial structures, connective tissue adhesions to adjacent tissues, as
well as other muscles (Burkholder, 2007; Huijing & Baan, 2001).
The ultimate aim of biological research is to understand the importance of infor-
mation, specifically the inter- and intracommunication of cells and molecules, and
their role in the development of and modulation of structure. Signals are forms of
information bridging the external and internal environment(s) of a living organism,
for although individual units (cells, molecules) feature prominently in the construc-
tion of the whole (tissues, organs, organisms), it is the sum of these units plus the
local interactions (information) suggesting that the whole is greater than the sum of
its parts.
Biological systems express a degree of randomness. The inherent positive and
negative feedback loops are mechanisms of signal transduction defining adaptation.
Adaptation governs the function of the cells considered as a form of a change or a
level detector, with the former being sensitive to permutations in the magnitude of
the stimulus and the latter to stimulus intensity (Mcburney & Balaban, 2009). The
change detector is a high-pass filter, sensitive to a high frequency, and a rapid stimu-
lus fluctuation, with the level detector being a low-pass filter, sensitive to a steady
low-frequency stimulus (Mcburney & Balaban, 2009). Common amongst both fil-
ters is the measuring and processing of information from the internal and external
environments decoded through in vitro and in vivo studies.
The magnitude, intensity, and rate of the transduced signal are core mechanisms
of cellular information processing mediated by the concentration of molecules,
themselves serving as signals (Uda & Kuroda, 2016). The signal generated holds
the potential of explaining any observed dynamic behaviour of living systems.
Understanding how this signal (information) is generated, stored, and used at the
various levels, from the macro to the micro, and vice versa, explains the complex
feedback and feedforward patterns of biological networks. These are a reflection of
underlying processes characterised by the information garnered by the low and high
frequency filters, providing a better understanding of the staggering complexity,
robustness, and versatility of the structure of living systems and their function
(Oltvai & Barabasi, 2002) in short, a manifestation of a specific integration of both
molecular and cellular information and its influence on form and function. Therefore,
the mechanical feedback or factors generated within and by the system is an impor-
tant determinant of organismic form and an expression of information generated by
the stiffness, stretchiness, and viscoelastic response of cells and tissues to a mechan-
ical load (Wainwright, 1988).
One can start from the analysis of elementary components of a phenomenon,
such as often studied by in vitro studies; however, to comprehend the reality of what
actually occurs, we need to comprehend the phenomenon in its entirety. According
to von Bertalanffy (Von Bertalanffy, 1968), one needs to observe from a higher level
and from a holistic perspective. The human body is a system, a coherent whole
consisting of a boundary distinguishing it from internal and external elements (Ng,
Maull, & Yip, 2009). Studying the specificity of individual biological molecules
involved in a structure or an activity is important, for complex structures and activi-
ties cannot be explained in isolation, since their components are frequently involved
in many different processes (Van Regenmortel, 2004). Therefore, interest in this
project is concerned with how the magnitude of a signal (information) in the form
of a stretch (external force) influences the macroscopic and microscopic elements of
the body. An understanding of the interrelationship(s) of the various components,
from the tissues to the muscle cell, provides a better understanding of how stretch-
ing may influence the structure and function of the body and its relevance to inflam-
mation and the inflammatory response.
Muscles, Tendons, and MTU
Muscle
Skeletal muscle is a composite of connective tissue, nerves, blood vessels, and pri-
marily contractile material, classified as either smooth and striated (cardiac and
skeletal) (Gillies & Lieber, 2011). This dynamic tissue that undergoes a significant
degree of mechanical strain and cellular deformation with each contraction is a
study of the interplay between chemical and mechanical inter-tissue signalling
(Gumerson & Michele, 2011). To preserve its normal function, the skeletal muscle,
which generates force and routinely undergoes cell shortening required for
movement, limits mechanical cellular injury by adapting to changing workloads,
thus ensuring a balance between degeneration and regeneration (Gumerson &
Michele, 2011).
Skeletal muscle consists of bundles of cells anatomically and mechanically
arranged in parallel, with each cell functioning independently and the total force
generated being a sum of the forces of each cell (Goldstein, Schroeter, & Michael,
1991). It is a highly specialised structure responsible for transforming chemically
stored energy into mechanical work, with the energy provided being in the form of
adenosine triphosphate (ATP) (Adams & Schwartz, 1980). This function is depen-
dent on the efficient coordination and integration of several subcellular biological
networks responsible for the contraction, shortening, and development of tension
(Smith, Meyer, & Lieber, 2013).
All skeletal muscles have adaptive potential, capable of modifying their structure
in response to environmental changes, such as altered patterns of activity, pathologi-
cal processes, metabolic conditions, and ageing (Bruton, 2002). When skeletal mus-
cle is not recruited to generate force and movement, it is normally found in a relaxed
state (Goldstein et al., 1991). The muscle possesses a remarkable ability of repair or
regeneration following damage via an effective cellular repair system (Philippou,
Maridaki, Theos, & Koutsilieris, 2012). This is stimulated in order to recover nor-
mal structure and function while preventing loss of mass since skeletal muscle is
considered to be an irreversible postmitotic tissue lacking an ongoing cell replace-
ment (Decary et al., 1997; Goldspink, 2005; Philippou et al., 2012). Skeletal muscle
has two specialised junctional regions, the MTU and neuromuscular, with the for-
mer referring to the attachment of the muscle to the tendons and the latter to the
innervation of the muscle by motor neurons.
Isometric, concentric, and eccentric activity are the three major muscular actions
resulting from both the muscle’s active and passive components (Knudson, 2007).
Isometric refers to an activity where the muscle length does not change, with both
concentric and eccentric describing the shortening and lengthening of the muscle,
respectively. Active and passive tension defines the length-dependent properties of
the muscle strongly related to stretching (Knudson, 2006). The interaction of each
suggests that exercise interventions, such as stretching, has a complex effect on
skeletal muscle dependent on the interaction of the tissues and the nature of the
training stimulus (Knudson, 2006).
Although much has been learned by observing muscle in response to various
activities (i.e. weightlifting, running, sprinting) and forces (i.e. compression, ten-
sion, bending, rotation, shearing, and gravity), to gain a better understanding and
insight into and about the mechanism(s) involved during force generation or its
response to stretching, it is necessary that we take the system apart. This reduction-
ist approach, with use of in vitro studies and assays, continues to contribute to the
study of the interaction of the filament systems of striated skeletal muscle (endosar-
comere) and its relationship and integration with the multitude of myofibrillar pro-
teins (Ackermann et al., 2011; Batters, Veigel, Homsher, & Sellers, 2014). As
discussed above an in vitro approach enables the simplification of complex objects
making them easier to understand.
Muscles are composed of tubular cells called myocytes containing many chains
of myofibrils and cylindrical structures, extending the complete length of the myo-
cyte. The streamlined cylindrical shape of the myofibril is important for transmis-
sion of forces, for cylinders are designed to provide a passage for the flow of
information from one place to another (Oiwa & Manstein, 2007; Volk, 1995). This
structural design ensures a fundamental requirement of all cells, their ability to
interpret, convert, and convey information gathered from their immediate environ-
ment. To accomplish such a task, several systems have been designed in nature. The
G-protein family, responsible for processing and converting information about
membrane shear stress (Frangos, Mcintire, & Eskin, 1988) and the integrin recep-
tors, sensing the surface tension of the cell through ligand binding (Ruoslahti &
Pierschbacher, 1988). In addition, there are the strain activated ion channels which
alter conductance when deformed (Guharay & Sachs, 1984), and the cytoskeletal
network (microfilaments, microtubules, intermediate filaments), responsible for
causing a conformational change in proteins and a deformation of the nucleus, alter-
ing biological activity (Maniotis, Chen, & Ingber, 1997; Palmisano et al., 2015). All
of these will be discussed further below.
The myofibril provides a scaffold for the spatial distribution of proteins respon-
sible for integrating force production and transmission (Sanger et al., 2005). The
smallest repeated segment of the myofibril, the sarcomere, is the fundamental unit
of muscle contraction dominating the anatomy of both skeletal and cardiac striated
muscle (Batters et al., 2014; Lange, Ehler, & Gautel, 2006; Sanger & Sanger, 2001)
(Figs. 2.2 and 2.3). A wide range of sarcomere lengths exist suggesting that no sin-
gle sarcomere length can be used to explain all muscles; however, what is known to
determine sarcomere length is the function of the muscle itself (Lieber, Roberts,
Blemker, Lee, & Herzog, 2017). Despite the fact that sarcomeric lengths have a
preferred operating range, published work by William and Goldspink suggesting
Fig. 2.2 The sarcomere. (Published with kind permission of copyright © Nikos C. Apostolopoulos
2018. All rights reserved)
Fig. 2.3 The sarcomere depicting the four filaments (in red). (Published with kind permission of
copyright © Nikos C. Apostolopoulos 2018. All rights reserved)
that sarcomeres adapt to a new length by adjusting the serial sarcomere numbers
(i.e. increase or decrease) has been questioned (Williams & Goldspink, 1978a,
1978b). A study by Takahashi et al. demonstrated that although serial sarcomere
numbers increased initially at the MTU 1-week postsurgical tensioning procedures,
when the MTU was stretched beyond its “physiological point”, this was followed by
a rapid decrease in sarcomere numbers over the remaining 8-week period, despite
sarcomere length being constant (Takahasi, Wand, Marchuk, Frank, & Lieber,
2010), suggesting that adaptation of the sarcomere of skeletal muscle is extremely
plastic.
The sarcomere is the largest macromolecular complex known, assembled from
many protein subunits organised in a highly specific way into filamentous (myosin,
actin, titin, and nebulin) and anchoring (Z-disc, M-band) structures responsible for
the contractile process (Gautel, Mues, & Young, 1999), and of cytoskeletal elements
and their relative proteins (Fig. 2.3). Although the sarcomere specialises in force
generation, its fundamental activity and response to a mechanical force (i.e. stretch-
ing) is dependent upon the correct positioning of hundreds of proteins located
within, as well as on its periphery. These cytoskeletal elements consist of both
dynamic and passive components involved in the transference of force but, more
importantly, the relaying of information from one sarcomere to another. These

proteins play both structural and regulatory roles and are responsible for connecting
the sarcomere to the plasma membrane and from there to the ECM. At this cellular
and organelle level, the exosarcomeric network, the intermediate filament lattice of
the cytoskeleton, envelopes the sarcomere linking the Z-disc and M-lines to the
membrane skeleton (costamere), mitochondria, nuclei, and sarcoplasmic reticulum
(Barral & Epstein, 1999; Sanger, Sanger, & Franzini-Armstrong, 2004; Wang,
Mccarter, Wright, Beverly, & Ramirez-Mitchell, 1993) (more discussed in sections
below). It is believed that in muscle cells, this intermediate filament network is force
bearing (Lazarides, 1980; Wang et al., 1993).
In addition to the exosarcomeric, the endosarcomeric network exists consisting
of four filaments: the thin and thick comprised mainly of the actin and myosin pro-
teins, respectively, as well as the third and fourth filaments, represented by the large
myofibrillar extensible titin and the inextensible nebulin proteins, respectively
(Kruger, Wright, & Wang, 1991; Tskhovrebova & Trinick, 2003) (Fig. 2.3). Both
the exo- and endosarcomeric network systems, and their associated proteins, are
responsible for the synchronous response and conversion of the microscopic con-
tractile process at the sarcomere leading to a macroscopic action.
The unique cross-striated appearance of striated muscle, first reported in 1840 by
Bowman (Bowman, 1840), arises as a result of the transverse alignment of sarco-
meric striations of neighbouring myofibrils (Wang & Ramirez-Mitchell, 1983). The
maintenance of this alignment is attributed to a filamentous bridge between the
M-line and Z-disc and the intermediate filament (Lazarides, 1980, 1982; Wang &
Ramirez-Mitchell, 1983). The alternating light (isotropic) and dark (anisotropic)
bands are direct and precise alignments of the four filament systems, the lateral
boundaries of the sarcomere defined by the Z-disc, and the intermediate filament of
the exosarcomere (Clark, Mcelhinny, Beckerle, & Gregorio, 2002; Rui, Bai, &
Perrimon, 2010).
The Filament Systems of Skeletal Muscle
Since the groundbreaking work of Hugh Huxley (Huxley, 1969; Huxley & Hanson,
1954), detailed investigations have been conducted of the properties of myofibrillar
proteins exploring the molecular basis for muscle contraction. What has been
uncovered is that the dynamic interaction responsible for the assembly of myofibrils
is dependent upon the coordinated integration of myosin, actin, and associated pro-
teins, as well as other cytoskeletal elements.
As mentioned above, four filaments comprise the sarcomere. The parallel arrays
of the thin filaments spanning the I band (light region), overlapping with the thick
filament in the A-band (dark region), and their affiliated proteins and protein com-
plexes, all responsible for the generation of active force and the subsequent perfor-
mance of mechanical work (Rui et al., 2010).
Situated in between two I-bands is the Z-disc, with the M-line located in the
middle of the A-band (Fig. 2.2). Both these transverse structures are important
regions, for the thin and thick filaments cross-link in the Z-disc and M-line,
respectively. This biological design serves an important structural and functional
role maintaining the integrity of the sarcomere during contraction and stretching by
establishing a precise arrangement of the filaments (Katzemich et al., 2012). The
amount of overlap is determined by the respective length of these filaments, in par-
ticular the thin filament. Research has uncovered that the length of the thick filament
is constant (1.6 μm); however the length of the thin filament fluctuates from~1.0 to
1.3 μm (Burkholder, Fingado, Baron, & Lieber, 1994; Littlefield & Fowler, 2008).
The extent of overlap between the thin and thick filament determines the sarco-
mere’s force-generating capacity as well as the physiological requirement of the
muscle (Burkholder et al., 1994). For instance, short thin filaments are associated
with a reduction in force due to a decrease in the overlap with the thick filament
(Granzier, Akster, & Ter Keurs, 1991; Ottenheijm & Granzier, 2010). In addition, it
has been observed that the M-line is more prone to distortion under stress compared
to the Z-disc (Agarkova & Perriard, 2005; Gautel, 2011; Linke, 2008).
Titin and nebulin form the third and fourth filaments, with titin spanning half the
sarcomere and nebulin traversing the full length of the thin filament, with both play-
ing an important role in sarcomere organisation, strength, and development (Clark
et al., 2002; Meyer & Wright, 2013) (Fig. 2.3). For a detailed review of the filament
system(s), their associated proteins, and the Z-discs and M-line of the sarcomere,
and cytoskeletal proteins, the reader is directed to the end of the chapter to the sug-
gested “Further Reading” section. The material in this section provides a brief over-
view of the structure and important components of the sarcomere in order to provide
an understanding of how these structures relate to one another and how stretching,
specifically the magnitude of stretching, may be responsible for causing an inflam-
matory response.
Thin Filament (Actin, Troponin, Tropomyosin, and Tropomodulin) (Fig. 2.3)

The thin filament consists of actin comprised of two twisted α helices associated
and affiliated with the regulatory protein complex consisting of tropomyosin
and troponin (Kostyukova, 2008; Rui et al., 2010; von der Ecken et al., 2015;
Wakabayashi, 2015). In addition, tropomodulin, which requires tropomyosin for
optimal function, is a pointed end thin filament capping protein associated with the
thin filament and nebulin (discussed below), responsible for maintaining the final
length of the thin filament, preventing it from elongating or shortening (Mcelhinny,
Kolmerer, Fowler, Labeit, & Gregorio, 2001; Ottenheijm & Granzier, 2010). It
also inhibits the depolymerisation and polymerisation of the actin monomers
(Kostyukova, 2008).
Tropomyosin and troponin are attached to the actin backbone at regular intervals
and are important for regulating contraction and the giant protein nebulin (Gordon,
Honmsher, & Regnier, 2000; Horowits, Luo, Zhang, & Herrera, 1996; Wang &
Wright, 1988; Zot & Potter, 1987). In skeletal muscle, tropomyosin forms a
continuous ropelike structure over a head-to-tail association strengthened by tropo-
nin (Schutt & Lindberg, 1992). Troponin, which binds to the tropomyosin molecule
is responsible for regulating the motion of tropomyosin on the thin filament (White,
Cohen, & Phillips, 1987). This relationship between tropomyosin and troponin is
very important for the generation of maximal force imparted on the Z-disc, for
tropomyosin relative to actin is an inextensible parallel component responsible for
summing up all the individual forces developed over the length of the actin filament
in the overlap zone between actin and myosin (Schutt & Lindberg, 1992).
The tension developed at the sarcomere is a manifestation of the helical segment
of the actin filament determined by its attachment to both the tropomyosin at one
end and to the myosin head on the other, with contraction occurring when the actin
segments pull on tropomyosin while being anchored via cross bridges to the thick
filaments (Schutt & Lindberg, 1992). The actin-tropomyosin-troponin filament pro-
tein complex is responsible for (a) the calcium (Ca2+)-dependent movement of tro-
ponin and tropomyosin on actin filament, since these two proteins are essential for
Ca2+ regulation (Gordon et al., 2000; Szczesna & Potter, 2002; Wakabayashi, 2015),
and (b) the interaction site(s) with myosin (Barua, Winkelmann, White, & Hitchcock-
Degregori, 2012; Borovikov & Gusev, 1983; Kad, Kim, Warshaw, Vanburren, &
Baker, 2005). This complex is anchored in the Z-disc extending towards the middle
of the sarcomere interdigitating with the thick filaments in the A-band region
(Fig. 2.2).
Thick Filaments (Myosin)

Myosin is the principle protein of the bipolar thick filament and, in association with
several other proteins, forms a remarkable and precise network for the purpose of
contraction in skeletal muscle (Adelstein, 1983). The associated proteins of the
thick filament are critical in mediating sarcomeric protein interaction, such as thick
filament assembly and regulation of muscle contraction (Barral & Epstein, 1999;
Clark et al., 2002; Kenny, Liston, & Higgins, 1999). Myosin provides the force
necessary to drive muscle contraction but is also important in the structural forma-
tion of the thick filament (Vikstrom et al., 1997). It is one of the three major classes
of molecular motors, the other two being kinesin and dyneins, with all three driven
by the hydrolyses of ATP (Llinas, 2015). Unlike kinesin and dyneins which interact
with the microtubule cellular tracks, myosin networks with actin filament tracks
play both a structural and enzymatic role in muscle contraction and intracellular
motility (Llinas, 2015; Lowey & Trybus, 1995; Oiwa & Manstein, 2007). Myosin
consists of 18 subfamilies, with myosin II, predominantly associated with skeletal
muscle (Lutz & Lieber, 1999; Oiwa & Manstein, 2007).
The highly asymmetric myosin II protein is comprised of two dimerised myosin
heavy chain (MHC) molecules with each forming a head and neck domain, inter-
twining with its neighbour to form an extended α-helical coiled-coil tail (Clark,
Langeslag, Figdor, & Van Leeuwen, 2007; Craig & Padron, 2004; Rayment et al.,
1993). Each myosin heavy chain consists of two subunits, the heavy and light mero-
myosin, with the head and neck being part of the former and the tail part of the light
meromyosin (Clark et al., 2007; Lutz & Lieber, 1999) (Fig. 2.3). Both the head and
neck are referred to as catalytic and regulatory domains (Borejdo, Ushakov, &
Akopova, 2002), with the former consisting of the NH2 terminal responsible for
binding to actin exhibiting an actin-activated ATPase activity (Schutt & Lindberg,
1992). The neck consists of the two myosin light chain molecules, the essential and
regulatory (Borejdo et al., 2002; Trybus, 1994), with these molecules functioning as
a lever arm for force production (Clark et al., 2002; Craig & Padron, 2004; Krendel
& Mooseker, 2005).
The ordered arrangement of myosin heads consists of an inherent periodicity of
approximately 14.3 nm intervals on a helical array that repeats at every 42.9 nm
(Trinick, 1996; Xu et al., 1999). The structure of the backbone of the thick filament
consists mainly of staggered, closely packed myosin tails running parallel to the
filament axis and is responsible for assembling into a bipolar filament (Adelstein,
1983). Assembly of the thick filament is very much dependent upon the interaction
between the rod segments of the myosin (Craig & Padron, 2004). According to
Crick’s “knobs-into-holes” model, stability for two α helices into a coiled coil is
exhibited by an electrostatic arrangement by the strong attraction of the hydropho-
bic residues in the core of the coil with hydrophilic residues lying on the surface
(Chew & Squire, 1995; Crick, 1953; Mclachlan & Karn, 1982). This charge distri-
bution within the myosin tail accounts for the fundamental periodicities associated
with the myosin filaments (Craig & Padron, 2004; Offer & Sessions, 1995).
Third and Fourth Filaments and Obscurin

Within the myofibril are found three of the largest proteins in the body, titin, nebu-
lin, and obscurin, with titin and nebulin comprising the third and fourth filaments of
the endosarcomere. Both titin and nebulin are integral components of the sarco-
mere, with obscurin intimately surrounding them, primarily at the level of the Z-disc
and M-line during myofibrogenesis and the M-line in adult myocytes (Bang et al.,
2001; Kontrogianni-Konstantopoulos, Jones, Van Rossum, & Bloch, 2003; Young,
Ehler, & Gautel, 2001).
Mechanical stability of the muscle is maintained by the presence of the extensi-
ble titin and the inextensible nebulin filaments, with both located in the gap region
between the thick and thin filaments (I- and M-band) and from the ends of the iso-
lated thick filament (Figs. 2.2 and 2.3). Besides mechanical stability, these endosar-
comeric filaments are responsible for maintaining the assembly characteristic of the
sarcomere (Funatsu, Higuchi, & Ishiwata, 1990; Granzier & Labeit, 2005).
Titin (Connectin) (Third Filament): The Titan Amongst Proteins

Titin, a force-bearing protein, is the largest polypeptide in the body, a giant myofila-
ment, whose name is derived from the Titan gods of Greek mythology, who pos-
sessed great strength and importance (Luft, 2006). Titin, once considered to play
only a structural role by acting as a scaffold, ensuring the continuity of the myofi-
brils of striated muscle, has now emerged as a candidate for a controller of filament
assembly, since it traverses from the Z-disc to M-lines (Furst, Osborn, Nave, &
Weber, 1988; Maruyama, Kimura, Ohashi, & Kuwano, 1981; Trinick, 1996). Titin’s
dynamic nature suggests it has an important role in intracellular signalling and the
passive mechanical properties of the sarcomere (Granzier & Labeit, 2004; Labeit
et al., 2003; Miller et al., 2003; Nishikawa et al., 2012; Tskhovrebova et al., 2010).
Its incorporation into the M-line network suggests a role as a stress sensor since the
M-line is believed to limit longitudinal misalignments of the thick filament during
contraction, responsible for making the position of the thick filaments unstable
(Agarkova & Perriard, 2005).
Titin is responsible for the assembly of myofibres with its deletion from the sar-
comere resulting in a total loss and disruption of the myofibril assembly, despite the
presence of other sarcomeric proteins (Van Der Ven, Bartsch, & Gautel, 2000;
Young et al., 2001). Spanning half the sarcomere, with its NH2 and COOH termini
in both the Z-disc and M-lines (the middle of the A band), respectively (Furst et al.,
1988; Labeit, Gautel, Lakey, & Trinick, 1992; Squire, 1997; Tskhovrebova &
Trinick, 2001), the NH2 terminal forms an elastic connection between the end of the
thick filament and the Z-disc in the I-band region (Luther, 2009).
As an elastic connector, this assures adaptation to I-band length changes during
contraction but more importantly contributes to the generation of passive shorten-
ing/tension of the sarcomere during a relaxed state (no active force generated by
myosin cross-bridges) (Furst et al., 1988; Kontrogianni-Konstantopoulos,
Ackermann, Bowman, Yap, & Bloch, 2009; Labeit & Kolmerer, 1995; Maruyama
et al., 1981; Tskhovrebova, Houmeida, & Trinick, 2006) (Fig. 2.4). The I-band
Fig. 2.4 Titin relationship to Z-disc and M-line. (Published with kind permission of copyright ©
Nikos C. Apostolopoulos 2018. All rights reserved)
domain is the only region exhibiting elastic stretch dependence, observed to

approximately double in length during normal activity (Trinick, 1996; Wang,
Wright, & Ramirez-Mitchell, 1985). In addition, since titin-containing filaments are
centrally symmetric to the M-line, this is responsible for keeping the A band at the
centre ensuring balanced myosin-cross-bridge forces in both thick filament halves
during active contraction (Horowits, Kempner, Bisher, & Podolsky, 1986).
The titin protein is primarily composed of 244 repetitive and 17 unique non-
repetitive domains, with the former consisting of 112 immunoglobulin and 132
fibronectin-III superfamily domains (~90% of its mass) (Gautel, 1996; Labeit et al.,
1992). The 17 distinct motifs, accounting for ~10% of titin’s mass, are comprised of
phosphorylation sites, a 28–30 residue proline-glutamate-valine-lysine (PEVK)
motif found in the I-region and a serine/threonine kinase domain located at the
COOH terminal in the M-line (Fig. 2.4) (Granzier & Labeit, 2005, 2006; Greaser,
2001; Sebestyen, Fritz, Wolff, & Greaser, 1996). The physiological force levels
responsible for maintaining the structural integrity of the sarcomere, specifically the
passive muscle force, are determined by the PEVK (Linke, Ivemeyer, Mundel,
Stockmeier, & Kolmerer, 1998; Trombitas et al., 1998). The repetitive domains
(immunoglobulin and fibronectin-III) provide binding sites for many diverse pro-
teins within the myofibre, including myofibrillar and membrane components, sig-
nalling molecules responsible for integrating mechanical and contractile activity
with regulatory mechanisms controlling both metabolism and gene expression
(Kontrogianni-Konstantopoulos et al., 2009).
Nebulin (Fourth Filament): The Uncertain Protein

Nebulin, another myofibrillar protein, received its name from the word nebulous
because of its unknown function which was eventually revealed in part by the gen-
eration of nebulin knockout mice models (Ottenheijm & Granzier, 2010). These
models exposed the important role that nebulin played in regulating contraction and
the overlap of the thin-thick filaments (Ottenheijm & Granzier, 2010). During the
stretching of the sarcomere, nebulin, located parallel to the actin filaments, remains
relatively fixed to these filaments (Horowits et al., 1996). This adherence is due to
nebulin being composed of ~185 α-helical modular repeats that are each ~35 amino
acids in size (McElhinny et al., 2001), with each repeat being a potential actin-
binding motif (Witt et al., 2006).
Nebulin functions as a “molecular ruler”, defining the length of the thin filament,
since a single nebulin polypeptide spans the length of actin (Horowits et al., 1996;
Pfuhl, Winder, & Pastore, 1994). Two studies with nebulin knockout mice observed
the constant length of the thin filament with nebulin present, with the length decreas-
ing in mice deficient of nebulin (Bang et al., 2006; Witt et al., 2006). This relation-
ship suggests that when sarcomeres are subjected to overstretch, both filaments,
actin and nebulin, act in a coextensive relationship in situ, since no change in length
is observed of the actin filament (Kruger et al., 1991). The length of the thin filament
is important for determining the amount of force generated by a muscle based on its
physiological requirement (Burkholder et al., 1994).
The NH2 terminus of nebulin is found at the pointed end of the thin filament
interacting with the capping protein tropomodulin (H-zone) (Labeit, Ottenheijm, &
Granzier, 2011; McElhinny et al., 2001), while the COOH-terminus is anchored to
the Z-disc via α-actinin (Horowits et al., 1996). Localised at the Z-disc, α-actinin, a
cytoskeletal actin-binding protein, forms a latticelike structure stabilising the mus-
cle contractile apparatus, as well as playing an important role in muscle contraction
(Sjoblom, Salmazo, & Djinovic-Carugo, 2008), for muscle fibres deficient in nebu-
lin develop less force (Labeit et al., 2011). Nebulin in conjunction with titin is an
important regulator of Z-disc structure, specifically in its assembly and width
(Gautel, Goulding, Bullard, Weber, & Furst, 1996). It plays a role in the transverse
linking of the myofibrils limiting the degree to which they can move transversely
during contraction or passive stretching, thereby preventing damage to the inter-
myofibrillar membrane systems (i.e. T-tubules and sarcoplasm reticulum)
(Ottenheijm & Granzier, 2010). In addition, nebulin is required to connect desmin,
the most abundant intermediate filament protein (discussed further below) in stri-
ated adult muscle to the Z-disc preventing a greater displacement of the Z-disc
which occurs during the stretch of the myofibre (Ottenheijm & Granzier, 2010;
Shah et al., 2002). In short, nebulin contributes to isometric force production, width
specification of Z-disc, and myofibrillar connectivity (Labeit et al., 2011).
Obscurin: The Concealed Protein

Obscurin (obscure) and nebulin (nebulous) share similar synonyms, vague and
ambiguous, for similar to nebulin, this novel titin-interacting protein was difficult to
characterise because of its somewhat low abundance, large size, and complexity
(Young et al., 2001). Obscurin, a multidomain protein concentrated on the peripher-
ies of the Z-disc and M-line of the sarcomere is composed of signalling domains
and adhesion modules arranged in tandem (Kontrogianni-Konstantopoulos &
Bloch, 2005). Both obscurin’s NH2 and COOH-terminal sequences are exposed at
the sarcomere surface, located to and restricted to the Z-disc and M-lines, an expo-
sure consistent with the distribution of the sarcoplasmic reticulum and T-tubules,
also located on the periphery of the sarcomere, suggesting that obscurin is the
anchoring mechanism for the specific localisation of this membrane system (sarco-
plasmic reticulum and T-tubules) (Kontrogianni-Konstantopoulos et al., 2003;
Kontrogianni-Konstantopoulos & Bloch, 2005). In addition, since obscurin binds to
titin and the sarcomeric myosin (Young et al., 2001), its termini are more accessible
to segments of the sarcoplasmic reticulum, the myofibrillar cytoskeleton, and the
surrounding myoplasm (Franzini-Armstrong, 1994; Kontrogianni-Konstantopoulos
et al., 2003; Kontrogianni-Konstantopoulos & Bloch, 2005; Young et al., 2001).
Obscurin, similar to titin and nebulin, fulfills the role of a “molecular ruler” as
suggested by its relationship with the sarcomere and the cytoskeletal components
(McElhinny et al., 2001). However, instead of determining the longitudinal
dimensions, continuous reticular labelling of obscurin in cross sections suggests
that it is associated with the diameter of the sarcomere enveloping the myofibrils at
the Z-disc and the M-line (Kontrogianni-Konstantopoulos et al., 2003). In animal
studies, removal of obscurin was associated with changes in the longitudinal

architecture of the sarcoplasmic reticulum and associated proteins, suggesting a
prominent role in aligning the sarcoplasmic reticulum with the myofibrils of striated
muscle cells. Besides affecting the integrity of the sarcolemma, obscurin is respon-
sible for reduced muscle exercise tolerance and with disturbances in the organisa-
tion of the ECM during skeletal muscle development (Lange, Perera, Teh, & Chen,
2012; Randazzo et al., 2013).
In conclusion, the role for titin and nebulin is the passive elasticity of the sarco-
mere, both longitudinally and laterally (Horowits et al., 1986), and the maintenance
of a static and dynamic stability during relaxation and contraction, by primarily
ensuring that the thick filaments are kept at symmetrical positions (Horowits et al.,
1986; Horowits & Podolsky, 1987). Both serve as scaffolds or length-regulating
templates of the thick and thin filaments, important for contraction (Kruger et al.,
1991). In addition, obscurin, an anchoring mechanism for the specific localisation
of the membrane systems (sarcoplasmic reticulum and T-tubules), is an integral and
important component for both the assembly of the membrane systems and the con-
tractile apparatus affiliated with and required for Ca2+ homeostasis (Kontrogianni-
Konstantopoulos et al., 2003, 2006; Young et al., 2001).
Z-Disc, Intermediate Filaments, and Costameres
The primary function of the proteins/structures of the cytoskeleton of the muscle

cell is to link, anchor, as well as tether structural components inside the cell, ensur-
ing a cooperative movement between the contractile and elastic components of the
muscle (Goldstein et al., 1991; Stromer, 1998). The Z-disc and affiliated proteins
continue this enigmatic role, with novel proteins and interactions continuously
being discovered (Faulkner, Lanfranchi, & Valle, 2001; Gontier et al., 2005; Sanger
& Sanger, 2008). The Z-disc is formed by the overlap of the thin filaments from
opposing half-sarcomeres, cross-linked to a stable network consisting of four pairs
of Z-filaments each comprised of α-actinin (Z-disc protein), associated with stable
cross-connections of thin filaments (Sorimachi et al., 1997; Stromer & Goll, 1972)
(Figs. 2.2 and 2.3).
Z-Disc
The Z-disc lies perpendicular to the myofibrils marking the end of the sarcomere,
and is considered a supramolecular structure linked to both muscle injuries and
atrophies (Faulkner et al., 2000; Gontier et al., 2005). It serves as an anchoring site
for the actin, titin, and nebulin protein filaments (Fig. 2.3) being the primary conduit
for the generation of force by contraction (Clark et al., 2002).
The intricate molecular architecture of the Z-disc, linking the contractile units of
the sarcomere in series, is central to collecting information of the mechanical force
generated by the interaction between the myosin and actin within the sarcomere
(Faulkner et al., 2001; Sanger & Sanger, 2008). In the past, the Z-disc was considered
as a passive structure of the sarcomere linking actin filaments of opposite polarity

(antiparallel) in subsequent sarcomeres for the successive transmission of force
generated in the myofilaments (Frank, Kuhn, Katus, & Frey, 2006; Squire,
Al-Khayat, Knupp, & Luther, 2005). However, changes in Z-disc structure associ-
ated with development of active tension in skeletal muscle suggest that it is a
dynamic structure involved in determining the mechanical properties of the muscle
(Goldstein et al., 1991).
Depending on their location, proteins associated with the Z-disc can be divided
into those partially located in the Z-disc and extending into sarcomere (i.e. titin,
actin, nebulin, MLP, myopodin, etc.) (Barash, Mathew, Lahey, Greaser, & Lieber,
2005; Barral, Hutagalung, Brinker, Hartl, & Epstein, 2002; Horowits et al., 1986;
Hutagalung, Landsverk, Price, & Epstein, 2002; Kruger et al., 1991; Linnemann
et al., 2010; Liu, Srikakulam, & Winkelmann, 2008; Mcelhinny, Kazmierski,
Labeit, & Gregorio, 2003; Powers, Nishikawa, Joumaa, & Herzog, 2016; Schutt
& Lindberg, 1992; Witt et al., 2006) and those located entirely within the Z-disc
(i.e. α-actinin, telethonin, CapZ, etc.) (Caldwell, Heiss, Mermall, & Cooper,
1989; de Almeida Ribeiro et al., 2014; Faulkner et al., 2000; Miller et al., 2003;
Rubtsov & Lopina, 2000; Torgan & Daniels, 2000, 2001; Wette, Smith, Lamb, &
Murphy, 2017; Yamashita, Maeda, & Maeda, 2003; Zhou et al., 2001) (Figs. 2.5
and 2.6). The numerous proteins of this highly organised 3-D structure form mul-
tiple protein complexes interacting with both extramyofibrillar and intramyofi-
brillar domains (Stromer, 1995) (Fig. 2.7). The extramyofibrillar consisting of
costameres and intermediate filaments will be discussed further below. The intra-
myofibrillar consists of the actin, titin, and nebulin filaments responsible for inter-
facing the thin and thick filaments between the Z-discs and the M-line. The
relationship and interaction of these protein complexes and domains are respon-
sible for two important attributes of the muscle cell cytoskeleton, its strength and
Fig. 2.5 Z-disc proteins partially located in Z-disc (associations and function). (Published with
kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)
Fig. 2.6 Z-disc proteins fully located in Z-disc (associations and function). (Published with kind
permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)
Fig. 2.7 Z-disc proteins, intra- and extramyofibril. (Published with kind permission of copyright
© Nikos C. Apostolopoulos 2018. All rights reserved)
its flexibility, accommodating changes in cell geometry in response to muscle

contraction or a mechanical stress (Stromer, 1998).
The architectural organisation of the Z-disc is responsible for actively coordinat-
ing various cellular signalling pathways (Gontier et al., 2005). For instance, the
width of the Z-disc depends upon a variety of factors such as muscle and fibre type
and the difference in actin filament overlap responsible for structural transitions
(Stromer, 1995). These transitions place a load on the mechanical properties of
skeletal muscle, observed during the response of the sarcomere to mechanical stim-
uli in the form of either a contraction or a stretch (Goldstein, Schroeter, & Sass,
1982; Stromer, 1995). The Z-disc maintains a certain form and spacing regardless
of an increase in load suggesting its ability to resist deformation with a passive
stretch (Goldstein et al., 1991). This structural criterion allows for the ease of trans-
mission of tension generated within the sarcomere to bones via the tendons, for the
Z-disc functions as an anchor, adjoining sets of thin filaments end-on-end in myofi-
brils (Goldstein et al., 1991).
Maintenance of form and spacing provides stability within the Z-disc lattice over
a long range of sarcomere lengths, an important feature in skeletal muscle, since it
operates over a range of sarcomere lengths, during extension and contraction
(Goldstein et al., 1991). In short, the muscle is better able to conform to change by
adjusting and adapting to any mechanical stress. Therefore, understanding the com-
plexity of the Z-disc proteins and their interactions is relevant to comprehending the
influence of a mechanical force (i.e. stretching) on the sarcomere and the subse-
quent biochemical response (i.e. mechanotransduction).
No sarcomeric protein is an “island” onto its own, for most have at least one if
not more binding partners forming a network to support, stabilise, and provide
cohesion to the myofibre (Faulkner et al., 2001). In addition, this network, in asso-
ciation with other proteins and protein complexes found within the extramyofibril
and the sarcolemma, forms a fundamental unit of response, a feedback-signalling
machine. This process allows for the proper and significant relay of information
from a mechanical stimulus (i.e. stretching) to the myofibre via the ECM, and
depending on its magnitude, this may influence the skeletal muscle tissue aversely
or favourably. An example of a negative disruption is eccentric contraction, an elon-
gation of the skeletal muscle in an activated state, responsible for damaging the
sarcomeres and impairing the excitation-contraction coupling (Agarkova et al.,
2003). Ultrastructural studies observed a characteristic change in the sarcomere pat-
terns associated with localised regions of overstretched sarcomeres exhibiting irreg-
ular and distorted Z-discs (Yeung, Belnave, Ballard, Bourreau, & Allen, 2002). The
stress on the sarcomere results in a breakdown to structures involved in the
excitation-contraction coupling (i.e. sarcoplasmic reticulum, T-tubules), itself linked
to a leakage of ions confirmed by measurement of intracellular Ca2+, resulting in
inflammation (discussed further below) (Balnave & Allen, 1995). For instance, pas-
sive stretching of mice skeletal muscle >50% of strain disrupted the sarcomere,
suggesting work done to stretch muscle is an indicator of the magnitude of injury
(Brooks, Zerba, & Faulkner, 1995). This response to the magnitude of a mechanical
stimulus (i.e. stretching) suggests that the intensity of the activity can alter the
morphology of the intra- and extramyofibrillar components initiating an inflamma-
tory response.
Besides the actin-myosin complexes, effective propagation of a mechanical sig-
nal (information) from the environment to the cytoskeleton, and the ensuing chemi-
cal response, is dependent upon several subcellular complexes. Apart from the
Z-disc and affiliated proteins, the other complexes consist of the cytoplasmic
intermediate filament (i.e. desmin) and the two major costamere complexes
(dystrophin-glycoprotein and the integrin-vinculin-talin).
Intermediate Filaments: The Mechanical Relay System

The depletion of thin and thick filaments from the myofibril with use of a high-salt
buffer results in a cytoskeleton composed predominantly of intermediate filaments
(Lazarides, 1980) (Fig. 2.8). These filaments form a dynamic cytoskeletal infra-
structure connecting the peripheries of each successive Z-disc as well as forming a
sleeve surrounding each myofibril (Wang & Ramirez-Mitchell, 1983). In cells sub-
jected to mechanical stress they play important roles as force mechanosensors and
mechanotransducers (Conover & Gregorio, 2011).
The intermediate filaments form a continuous network linking the cell mem-
brane to the cytoplasmic organelles and nuclear envelope thereby functioning and
acting as a mechanical relay system for the transmission of muscle tension
(Anderson, Li, & Goubel, 2001; Conover & Gregorio, 2011). This network is
responsible for diverse functions such as force transmission, mechanochemical sig-
nalling, cellular integrity, and the mechanical integration of the contractile actions
of the muscle fibre (Capetanaki, Bloch, Kouloumenta, Mavroidis, & Psarras, 2007).
The skeletal muscle intermediate filaments associate with the sarcolemma at struc-
tures termed “costameres”, which are present at the membrane overlying the Z-discs
of the sarcomere (Capetanaki et al., 2007).
Fig. 2.8 Intermediate filaments and costameres. (Published with kind permission of copyright ©
Costameres: The Achilles Heel

The relationship between the costameres and muscle is likened to the Greek heroes,
Hercules and Achilles, with the muscle responsible for performing impressive feats
of strength (Hercules) and the costamere, its Achilles heel (Ervasti, 2003).
Costameres, transverse elements of the sarcolemmal lattice, resemble riblike struc-
tures (“costa”, Latin for “rib”, and Greek “meros”, part) consisting of a doublet
band of densely clustered patches of vinculin (a cytoskeletal protein involved with
cell-cell and cell-matrix junctions) segregated into two rows flanking the Z-disc as
well as overlying the I band of the underlying sarcomere (Pardo, D’angelo Siliciano,
& Craig, 1983) (Fig. 2.8). The costameres circumferentially align in register with
the periphery of the Z-disc of the force generating myofibrils, physically coupling
the Z-disc to the sarcolemma (Ervasti, 2007). Costameric proteins identified and
associated with the costamere/Z-disc axis are responsible for converting a mechani-
cal stimulus to a change in both cell signalling and gene expression (Ervasti, 2003).
Whether the muscle is contracted or stretched, the periodicity of the costamere is
identical to the underlying sarcomere, with this relationship observed whether a
bundle of superficial myofibrils is staggered with respect to an adjacent bundle,
displaced, or discontinued (Pardo et al., 1983).
Costameres are composed of large complexes of integral and peripheral mem-
brane proteins linked to the contractile apparatus of the sarcomere by intermediate
filaments and to the ECM (Pardo et al., 1983). Besides playing a mechanical and
functional role for the transmission of force from the contractile apparatus to the
ECM, costameres protect the sarcolemma against contraction-induced damage
(Anastasi et al., 2008).
Although it is beyond the scope of this manuscript to describe the numerous
relationships and nuances formed between the sarcomere, the sarcolemmal struc-
tures (intermediate filament and costamere complexes), and the ECM, in response
to a mechanical signal, what is presented is a simple hierarchy of interaction. This
level of interaction is descriptive of a dynamic relationship and integration of
numerous proteins, and protein complexes, responsible for a signal transduction
cascade, specifically the transference and processing of information. Movement of
this communication is accomplished by a cell-wide system, a highway, composed
of key inter- and extramyofibrillar proteins responsible for the connectivity span-
ning the nuclear matrix to the ECM and beyond. This continuous network ensures
transduction of a mechanical signal from the environment, traversing and influenc-
ing the response(s) of the structures affected at both the macroscopic (i.e. muscles,
MTU, tendon) and microscopic (ECM, myofibrils, nucleus) levels. The result is a
biochemical response, a feedback to the mechanical signal, and a mechanotransduc-
tion process.
Communication with cells is made possible through a tensegrity architecture for
structural organisation (Ingber, 1997), the integrity of an object under tension, a
term coined by Buckminster Fuller, based on an object created by his student,
Kenneth Snelson (Connelly & Back, 1998). According to tensegrity, objects
maintain their structural integrity by achieving a balance between compression and
tension. Use of tensegrity to describe the relationship between the various levels of
tissues and cells in response to a dynamic external load (i.e. stretching) provides an
explanation for the hierarchy of interaction mentioned above. As the mechanical
signal passes from the macroscopic to the cellular environment, the cells sense and
transduce the force, accomplished by protein structures responsible for creating a
tensional force balanced by interconnected structural elements resisting compres-
sion within the cells of musculoskeletal tissues (Anastasi et al., 2006; Connelly &
Back, 1998). The result is the transduction of the force into changes of cellular
biochemistry and gene expression, mechanotransduction (discussed below).
The structural and regulatory proteins associated with this cell-wide system are
responsible for both active and passive load bearing within muscle sarcomeres
(Shah et al., 2004). Of the Z-disc proteins, titin, actin, and α-actinin play a promi-
nent role in establishing a connection with the ECM, with disruption of the interac-
tion between titin and α-actinin translating into an interruption within structure of
the Z-disc, since the amount of titin overlap coincides with the width of the Z-disc
(Gregorio et al., 1998). Titin is a Z-disc linking protein acting to specifically deter-
mine attachment of the α-actinin within the Z-disc creating sites for the cross-
linking of the thin filaments (Sorimachi et al., 1997). Α-actinin functions as a ligand
for titin enabling the cross-link of the antiparallel titin and thin filaments from
opposing sarcomere (Sorimachi et al., 1997) (Fig. 2.8), With the interaction of
α-actinin, titin, and actin contributing to the structural continuity of the sarcomere.
The establishment of a connection between the myofibril and the ECM is mani-
fested by a system of intermediary components acting between the Z-disc. The
structure and function of these intermediate filament proteins connect the contrac-
tile apparatus to the sarcolemmal cytoskeleton. The most predominant intermediate
protein is desmin, essential for maintaining the integrity of the myofibrils upon
stress (Li et al., 1997). Desmin is co-expressed with the proteins synemin and par-
anemin, forming copolymers typically localised around α-actinin-rich Z-discs
(Costa, Escaleira, Cataldo, Oliveira, & Mermelstein, 2004). This interrelationship
suggests formation of large intermediate filament protein networks creating a
greater opportunity for interaction with other structures (Capetanaki et al., 2007;
Schweitzer et al., 2001). Synemin which consists of binding sites for desmin,
a-actinin, and vinculin (quintessential costameric protein) plays an important func-
tion directly linking intermediate filaments to both the Z-disc and costameres
(Bellin, Huiatt, Critchley, & Robson, 2001).
Lack of desmin in skeletal muscle is associated with irregularities in the organ-
isation of myofibres (i.e. loss of alignment of myofibrils), Z-disc streaming, focal
degeneration, but more importantly a reduction of myofibril anchorage to the plasma
membrane at the costameres (Agbulut et al., 2001). Studies in mice lacking desmin
have observed that desmin-based intermediate filaments are important in linking the
Z-discs of superficial myofibrils to costameres at the sarcolemma (Stone, O’Neill,
Catino, & Bloch, 2005). In addition, desmin is essential for the nucleus to with-
stand any mechanical load associated with contraction, for an extensive loss of its
position occurs in absence of desmin during stretching of a passive fibre (Shah
et al., 2004). Through its high-affinity association with nebulin, desmin attaches to
the sarcomeres (Conover & Gregorio, 2011). Since desmin contains multiple
binding sites for nebulin, this allows for the preservation of the interfibrillar con-
nectivity at the Z-discs, the proper assembly of desmin intermediate filaments at the
Z-disc, influencing the architecture of the actin thin filament in myocytes (Conover
& Gregorio, 2011). This relationship between desmin and nebulin is crucial for the
stability and proper spacing of adjacent thin filaments required for the proper trans-
mission of the lateral force from Z-disc to Z-disc (Conover & Gregorio, 2011).
Costameres, concentrations of ECM receptor complexes, refer to a connection
site made between the contractile apparatus (myofibrils) and the sarcolemma. They
are involved in the transmission of and sensing of mechanical stress associated with
muscles and are considered a “proteic” machinery associated with the sarcolemma,
signalling, transmembrane, and ECM proteins (Anastasi et al., 2008; Gumerson &
Michele, 2011) (Fig. 2.9). Costameres are considered bands of vinculin, encircling
muscle fibres, repeating along its length with a periodicity corresponding to subja-
cent sarcomeres (Craig & Pardo, 1983). They have many characteristics in common
with the cell-ECM type of adherens junction, in particular focal adhesion com-
plexes, since they function as a cellular communication unit, a signalling highway
tethered between the Z-disc, a hotspot for muscle cell signalling and the ECM
(Anastasi et al., 2009; Burridge & Guilluy, 2016; Peter, Cheng, Ross, Knowlton, &
Chen, 2011). In addition, they are associated with the protection of the sarcolemma
against contractile damage, for the membrane-associated costameres are coupled
physically to underlying sarcomeres, widening and narrowing in concert with
underlying I band in stretched and contracted muscle (Craig & Pardo, 1983; Peter
et al., 2011).
Two protein complexes concentrated at the costamere and registered to the Z-disc
of the sarcomere are, the dystrophin-glycoprotein and the integrin-vinculin-talin
complex. Both are responsible for regulating the interaction between the cytoskel-
eton and the ECM (Anastasi et al., 2004). The former complex, consisting of dys-
trophin (an elongated cytoskeletal protein), dystroglycan, and the sarcoglycan
transmembrane subcomplex, plays a critical role between the ECM-cell interaction
(Anastasi et al., 2003). Dystrophin, linking the submembrane actin and the intermediate
Fig. 2.9 Costamere associations. (Published with kind permission of copyright © Nikos

filament is important for the transmission of the force of contraction across the
sarcolemma to the extracellular structures and maintenance of the architecture of
skeletal muscle (Ervasti & Campbell, 1993; Peter et al., 2011). The dystroglycan
and sarcoglycan complexes link the signal transducing units of the ECM to the
myofibrillar contractile elements (Anastasi et al., 2004; Ohlendieck, 1996).
Dystyroglycan, generated from a single gene is cleaved into two proteins, the
peripheral α-dystroglycan and the transmembrane β-dystroglycan (Michele &
Campbell, 2003), with the former binding to α-laminin, a component of the ECM,
and the latter, connecting the cytoskeleton to the ECM by binding to dystrophin
(Campbell, 1995), completing a pathway responsible for transferring a mechanical
signal between the ECM and cytoskeleton. Closely associated to the dystroglycan
complex is the multi-member sarcoglycan complex, consisting of six genes, α-, β-,
γ-, δ-, ε-, and ζ-sarcoglycan, of which the α- and γ- are associated with skeletal
muscle (Anastasi et al., 2009). This transmembrane protein stabilises the interac-
tions of dystrophin and its associated proteins (dystroglycan with the ECM) (Hack,
Groh, & Mcnally, 2000). A marked reduction in dystrophin and the associated gly-
coproteins (dystroglycans and sarcoglycans) disrupts a critical linkage between the
sarcolemmal cytoskeleton actin and the ECM, compromising the integrity and flex-
ibility of the sarcolemma, affecting its mechanical stability during cycles of con-
traction and relaxation (Campbell, 1995).
The other complex, the vinculin-talin-integrin, regulates interactions between
the cytoskeleton and the ECM, by the sensing of forces (compression, tensile, shear,
etc.) from both an “outside-in” and “inside-out” signalling process. It is an integral
part of a myriad of tissues (muscle, tendons, bones, etc.) exposed to an ever-
changing mechanical stimulus generated by environmental forces and movement
(Atherton, Stutchbury, Jethwa, & Ballestrem, 2016; Dufort, Paszek, & Weaver,
2011). In fact, the sustained disruption of the tensional homeostasis of the cell and
tissues caused by a mechanical stimulus results in alterations in the ECM, serving
as a catalyst for either repair or harm. A major player coordinating this mechanore-
sponsiveness is vinculin, a mechanosensitive protein involved in the adaptation of
tissue to forces and the homeostasis of the actin cytoskeleton (Atherton et al., 2016).
It is governed and regulated by intracellular forces generated during force transduc-
tion in relation to talin (Atherton et al., 2016). Talin, a ubiquitous large focal adhe-
sion protein, is an essential structural link between integrin and the intracellular
machinery, activating integrin and coupling it with the actin cytoskeleton (Atherton
et al., 2015, 2016; Humphries et al., 2007). This process is reinforced by vinculin
(Atherton et al., 2015, 2016; Humphries et al., 2007), for the force-induced mechan-
ical unfolding of talin exposes recognition sites for vinculin, with its binding to talin
being essential for the process of mechanotransduction at the cell-matrix adhesion
site (Haining, Lieberthal, & Del Rio Hernandez, 2016). The importance of force in
this whole process was demonstrated by both in vitro (Ciobanasu, Faivre, & Le
Clainche, 2014) and in vivo (Margadant et al., 2011) studies. The former, composed
of substrate-bound talin and actomyosin, demonstrated that the isometric contrac-
tion of actin exposed the vinculin-binding sites of talin increasing the affinity of
vinculin to talin while decreasing talin refolding (Ciobanasu et al., 2014). Similarly,
the latter study, with use of fluorescence microscopy, observed that cyclical
stretching of talin by the contraction of actomyosin resulted in recruitment of vin-
culin (Margadant et al., 2011), highlighting the importance of the magnitude of the
force. Therefore, the mechanotransduction response of talin, its unfolding, and the
binding of vinculin, are important processes responsible for a greater stabilisation
and adhesion between the cytoskeleton and the ECM (Haining et al., 2016).
As indicated above, talin and vinculin, which are tethered at the costamere, are
localised at the site of the cell-matrix adhesion interacting with integrin (Peter et al.,
2011). Integrin, a major force-bearing adhesion receptor protein, plays a dynamic
critical role in the cell’s ability to sense and respond to the mechanics of its sur-
roundings (Puklin-Faucher & Sheetz, 2009). A key player in both cell signalling and
adhesion, it is an ideal candidate for mechanotransduction, since the application of
force stimulates its recruitment to the cell-matrix adhesion site (Galbraith, Yamada,
& Sheetz, 2002), with vinculin stabilising the integrin-talin-actin complex, reducing
focal adhesion turnover (Critchley, 2005). This binding of vinculin to talin in
response to the magnitude of a stretching force is pivotal for coupling integrin to the
actin cytoskeleton and the transmission of force from the matrix to the actin cyto-
skeleton (Critchley, 2005; del Rio et al., 2009).
Integrins, allosteric proteins, are transmembrane heterodimeric receptors of α/β
subunits (Green et al., 2011; Mayer, 2003), integrating the cells’ exterior (ECM)
with its interior (cytoskeleton), maintaining the integrity of the cytoskeletal-ECM
linkage (Barczyk, Carracedo, & Gulberg, 2010; Campbell & Humphries, 2011; Van
Der Flier & Sonnenberg, 2001). They play a crucial role as direct mechanotransduc-
ers, transmitters of force to other elements (Ross et al., 2013). To date 18α and 8β
chains have been discovered paired in a manner forming at least 24 receptors for
cell adhesion molecules (Mayer, 2003; Plow, Haas, Zhang, Loftus, & Smith, 2000).
Molecules involved in cell adhesion are responsible for binding cells together into
multicellular organisms and tissues, enabling the communication of cells and tis-
sues with their surroundings (Van Der Flier & Sonnenberg, 2001).
Integrins have evolved a highly responsive receptor mechanism, enabling the
bidirectional transmission of signals (“inside-out” and “outside-in”) between the
ECM and intercellular interactions (Anastasi et al., 2004; Burkin & Kaufman,
1999), which explains their involvement in various biological phenomena (i.e. tis-
sue repair and remodelling and cell migration) (Belkin et al., 1996). As receptors for
cell adhesion molecules, integrins create appropriate opportunities for mechanical
connectivity by bringing cells together to form tissues. Tissues are the instrument by
which cells communicate with the environment in response to a wide range of phys-
ical forces (Schwartz, 2010). How this is accomplished is a hallmark function of
integrins. Through the α/β pairing, a recognition for multiple extracellular ligands
occurs, with the primary function and purpose being cell adhesion, permitting trans-
mission of signal and cell-cell interactions.
Ligands are large multi-adhesive ECM molecules binding to integrins (Barczyk
et al., 2010). The α/β pairings of the integrin determine the ligand-binding specifici-
ties for the various ECM proteins (Anastasi et al., 2004). Most α subunits have been
paired with one or two β subunits. The β subunit, composed of three subfamilies
(β1, β2, and β3), is used to group the integrins, with β1 and β3 mediating cell-matrix
adhesions expressed by all cell types and β2 restricted to leukocytes (Green, Mould,
& Humphries, 1998). The β1 subfamily forms the largest group of receptors for
ECM proteins (Mayer, 2003). It is expressed in mammalian focal contacts, costa-
meres, the myotendinous junction, and the sarcolemmal membrane (Mayer, 2003).
The β1 subfamily features prominently in the binding of an integrin receptor to the
ligands of muscle of the ECM, the fibronectin (α5β1), laminin (α6β1 and α7β1), and
collagen (α1β1 and α2β1). The dynamic formation between the integrin and its
specific ligand is a requirement for the process of mechanotransduction. Because of
this integrin-ligand interaction, cell adhesion and its engagement with the ECM
induce integrin activation and intracellular signalling; nonetheless, with time, this
adhesion-induced integrin activation subsides with the cell reaching a new state of
equilibrium (Jalali et al., 2001). Since it is beyond the scope of the present manu-
script to describe the numerous combinations of α/β pairings and variations and
their ligand molecule(s), the reader is directed to articles at the end of the chapter.
In summary, every component of the mechanical linkage from the external envi-
ronment to the cell-matrix (ECM ligands, integrins, costameres, intermediate fila-
ments, etc.) and the cytoskeleton (sarcomeric proteins, Z-disc proteins, etc.) is
responsible for the mechanical properties of the muscle and its response to a
mechanical load. The associations developed between these proteins and protein
networks establish a hierarchy of interaction, an information-processing pathway
by which the cell communicates with the environment via cell adhesion. This
dynamic connection between the specific ECM ligands and the integrin-mediated
adhesions is critical for the transmission of a signal into the cell consisting of physi-
cal (rigidity, composition) and biochemical (extracellular protein ligands) informa-
tion, information generated by the ECM protein networks [ligands (collagens,
laminins, and fibronectin)]. This signal, a response to a physical displacement of the
protein networks by exogenous and physiological forces, influences the mechanical
connectivity within each tissue and cell (Paluch et al., 2015). This process, a key
inherent design of complex organisms, allows for the modulation of the mechanical
strength of tissues to match the force encountered (Schwartz, 2010). For instance,
physical stress, such as the stretching of a single force-bearing cytoplasmic mole-
cule in response to cyclical stretching alters the proteins’ unbinding kinetics, expos-
ing binding sites for other proteins (i.e. talin for vinculin) (del Rio et al., 2009;
Margadant et al., 2011). This response influences the cell-matrix interface in an
attempt for the cell to adapt and establish a new equilibrium (Margadant et al.,
2011). When cells and tissues are subjected to forces that are too high, the estab-
lished regulatory mechanisms involved in maintaining their integrity breaks down.
This loss in integrity is responsible for the release of cytoplasmic contents into the
surrounding tissue sending chemotactic signals (movement of a cell in response to
a chemical stimulus) into the tissue prompting the inflammatory response and the
recruitment of inflammatory cells to the site of damage (Clarkson & Hubal, 2002;
Elmore, 2007; Robertson, Maley, Grounds, & Papadimitriou, 1993).
Muscle Architecture
Although the microscopic structures of the sarcomere, the four myofilaments, the
Z-disc, and the affiliated proteins form the functional unit of the muscle, with sarco-
mere length strongly influencing force generation or elongation, the best predictor
of force generation of the muscle macroscopically, is muscle architecture (Lieber &
Friden, 2000). Relative to the axis of force generation, the arrangement of muscle
fibres within a muscle determines its force-velocity properties (Lieber, 1992;
Moreau, Simpson, Teefey, & Diamano, 2010).
Behaviour of muscle contraction in humans has been evaluated based on the
assumed in vivo function associated with joint(s) action. Since direct observation is
impossible, the assumption has been that action at the joint is a reflection of the
intrinsic characteristic of muscle fibres and how they are affected by anatomical
factors within the muscle and joint system (Bobbert, Ettema, & Huijing, 1990;
Powell, Roy, Kanim, Bello, & Edgerton, 1984; Trotter, 1993; Wickiewicz, Roy,
Powell, & Edgerton, 1983; Wickiewicz, Roy, Powell, Petrine, & Edgerton, 1984). In
short, the force-velocity relationship reported for the muscle is similar to the muscle
fibres (Fukunaga, Kawakami, Kuno, Funato, & Fukashiro, 1997). This assumption
is controversial for one cannot obtain information about the amount of shortening or
lengthening occurring during the movement of the joint (Fukunaga et al., 1997). For
instance, pennate muscles (unipennate, bipennate, multipennate), where muscle fas-
cicles are arranged diagonally, running at several angles relative to the muscle’s
force generating axis, make it very difficult to determine the architecture of the
muscle during joint movement (Bobbert et al., 1990; Roy & Edgerton, 1992; Trotter,
1993) not to mention that it is almost impossible to describe the behaviour of the
MTU from joint movement (Fukunaga et al., 1997).
Regardless of this controversy, developments in imaging techniques (i.e. ultraso-
nography and MRI) have made it possible to determine function of the muscle-
tendon complex based on the structural alignment of the muscle fibres (Fukunaga
et al., 1992; Hensriksson-Larsen, Wretling, Lorentzen, & Oberg, 1992; Kawakami,
Abe, & Fukunaga, 1993; Kawakami, Abe, Kuno, & Fukunaga, 1995). These obser-
vations propose that orientation of the muscle fascicle suggests direction of the
muscle’s pull and its strength (Lieber & Friden, 2000). If fibres extend parallel to
the muscle’s force-generating axis, they possess a longitudinal architecture (i.e.
biceps brachii). If they are running at a fixed angle (0–30°), they are classified as
unipennate (i.e. vastus lateralis) and multipennate referring to fibres running at sev-
eral angles relative to the muscle’s force-generating axis (i.e. gluteus medius). This
structure-function relationship defines force production and movement providing a
plausible explanation of the mechanical basis of muscle injury during movement
(Lieber & Friden, 2000). According to Gans et al., the architecture of the fibre(s)
and how they are attached to each other influences force generation, performance,
and function (Gans & Abbot, 1991).
Tendon
As suggested by sections above, a complex dynamic interaction exists between the

cell and its physical microenvironment. This interaction involves a set of pathways
between its surface (i.e. focal adhesions, integrins, intermediate filaments, cytoskel-
eton) and the nucleus, culpable of a biochemical response to a mechanical perturba-
tion (Goldmann, 2012). This pathway, integral to the cell’s ability to adapt in
response to an externally applied force (tension, compression, shear stress, stretch-
ing), results in the stimulation of biochemical pathways fundamental for tissue
development, homeostasis, maintenance, and repair (Banes et al., 1995).
Tendons are rich ECM connective tissue structures consisting of strong collagen
fibril arrays and a tenocyte (a fibroblast), found within the ECM (Subramanian &
Schilling, 2015). In response to a mechanical force, collagen and prostaglandins,
released by the tenocytes, characteristic cells in tendons and ligaments, modify the
composition and elastic properties of the ECM (Milz, Ockert, & Putz, 2009;
Valdivia, Vega-Macaya, & Olguin, 2017). According to Kjaer, these changes to the
ECM are responsible for the tendon’s ability to resist the mechanical load generated
during muscle contraction as well as form a functional attachment to the bone
(Kjaer, 2004; Milz et al., 2009).
Tendons, are a specialised load-bearing tissue whose cells are sensitive to
mechanical stimuli during loading. They adapt their ECM in an anabolic or cata-
bolic manner, influenced by the magnitude, frequency, direction, and duration of the
applied load(s) (Lavagnino et al., 2015). Tendons primarily function by transmitting
tensile load to the bone from the muscle, ensuring stability and a greater efficiency
in the movement of the musculoskeletal system (Gelberman, Goldberg, & An,
1988). It has been suggested that during eccentric contractions they act as mechani-
cal buffers protecting the muscle from damage (Latash & Zatsiorsky, 1993).
Structurally, the tendon is attached to the muscle at the MTU and to the bone via
the teno-osseous junction. This viscoelastic material, of limited elongation, consists
of dense wavy (zigzag) parallel fibres that change as the load increases, in a time-
dependent manner. The limited elongation of the tendon is critical for its non-linear
viscoelastic response to applied loads (Summers & Koob, 2002; Woo & Buckwalter,
1988). As with all biological systems exposed to mechanical stress, the properties of
the tendon are highly dependent on their structure (tendon fibres) and cellular
organisation, particularly the orientation and composition of its ECM (PG, glyco-
proteins, elastin, and water) (Duenwald, Vanderby, & Lakes, 2009; Wren, Beaupre,
& Carter, 2000). Primarily composed of water, the ECM consists of type I collagen
(~65–80% dry weight) and elastin (1–2% of dry weight) (Lavagnino et al., 2015).
Tendons are arranged in a structural hierarchy, with each level consisting of
inherent tensile properties progressing from type I collagen (microscopic) m olecules
to collagen fibrils, that are assembled into fascicles, characteristic of the crimped
pattern, and finally into the tendon fibre (macroscopic) (Kastelic, Galeski, & Baer,
1978; Summers & Koob, 2002). The structural arrangement and the inherent
mechanical property of collagen is responsible for the main physical characteristics
of the tendon (Lavagnino et al., 2015). As the magnitude of force or load is increased,
the collagen’s ability to form covalent intermolecular and intramolecular cross-
links, inhibiting sliding between adjacent fibres and fibrils, allows the tendon to
withstand high tensile stress (~50 to 150 MPa) (Lavagnino et al., 2015; Woo &
Buckwalter, 1988). The linear region of the stress-strain curve of a tendon is asso-
ciated with the extension of the collagen fibres in the direction of traction, com-
bined with a sliding of the fibrils in their ECM (Goulam Houssen, Gusachenko,
Schanne-Klein, & Allain, 2011). Once the load is removed, the majority of changes
caused are recoverable, with the time required for recovery after unloading being
greater than the time required during the loading period of the tendon (Gelberman
et al., 1988).
The structure of the tendon is poorly suited to withstand compressive loading
(Wren et al., 2000), with chronic stress loads or overuse injuries causing multiple
micro-traumatic events, disrupting its internal structure, and is responsible for the
degeneration of the tendon cells and matrix (Kraushaar & Nirschl, 1999). An
in vitro study examining the effects of stretch on a rabbit tendon fibroblast cell
revealed that the mechanical load (stretch), and the subsequent biochemical
response (pro-inflammatory cytokine IL-1β), was responsible for degenerative
changes (Archambault, Tsuzaki, Heerzog, & Banes, 2002).
Myotendon Unit (MTU) (Fig. 2.10)
Referring to the function and architecture of the muscle, the tendon, and the MTU,
an appreciation is gained for the role each plays in relation to the stress or strain
developed both extrinsically and intrinsically. During intrinsic loading, the force
developed by the contractile filaments of skeletal muscle progresses through the
MTU to the tendon transforming the force from the muscle to the bone, creating
joint movement (Huxley, 1969; Mccall, Byrnes, Dickinson, Pattany, & Fleck, 1996).
Throughout this transition, the ECM is responsible for a functional link amongst the
three tissues, playing a fundamental role in the transmission of force and the main-
tenance of tissue structure of tendons, ligaments, bone, and muscle (Kjaer, 2004).
According to a comprehensive review, the ECM of both the tendons and intracel-
lular connective tissue is a dynamic structure adapting structurally and functionally
to mechanical load (Kjaer, 2004). In addition, the magnitude, frequency, and dura-
tion of forces on these tissues depend on their prior loading history (exercise, over-
use, disuse) and the composition of the ECM (age, disease, microdamage) (Tidball
& Quan, 1992).
The MTU found at either end of long cylindrical skeletal muscle fibres, is con-
sidered a significant component of the tension transmitting mechanism. It is charac-
terised by four separate ultrastructural domains connecting the actin filaments
and the associated cross-linking structures of the terminal sarcomere, with the
matrix occupying the space between the collagen fibres of the tendon (Tidball,
1984; Trotter, Eberhard, & Samora, 1983). Of the sarcomeric forces generated and
transmitted onto a tendon, a significant part of the force created is oriented parallel
Fig. 2.10 Myotendon unit. (Published with kind permission of copyright © Nikos

to the direction of the force ensuring that the junction is loaded in shear (Huijing &
Baan, 2001; Trotter et al., 1983). This force is generated by transmembrane pro-
teins, and not by the continuous bilayer of lipids (phospholipid, glycolipids, and
cholesterol) separating the myofilaments from the connective tissue filaments
(Trotter, Corbett, & Avner, 1981).
To facilitate this transmission by shear force, the MTU presents a complex geom-
etry, with the plasma membrane at each end of the muscle cell being highly folded
and invaginated, forming branch fingerlike cylindrical-shaped cell processes (Law,
1993; Trotter, Hsi, Samora, & Wofsy, 1985). Cylindrical shapes used in nature are
synonymous with a structural connection allowing for the transmission of forces, a
passage from one place to another (Volk, 1995). The cylindrical geometry of the
digit-like extensions greatly amplifies the interfacial area between the muscle and
connective tissue resulting in a decrease in stress at the muscle-tendon interface
(Tidball, 1984; Trotter, Samora, & Baca, 1985). A reduction in junctional surface
area is often accompanied by an increase in junction failure, suggesting that stresses
in pathological conditions are higher than normal resulting in a mechanical failure,
although the cells are less capable of force production (Tidball, 1984). Observations
of this interfacial folding by investigators have interpreted this folding as an

extension of the muscle cell into the tendon, with a complimentary extension of the
tendon into the muscle cell (Mackay, Harrop, & Muir, 1969; Tidball, 1983). In addi-
tion to the folded interface, transmission of force from one sarcomere to its serial
neighbour and eventually onto the MTU supports the viewpoint that the MTU is a
specialised tissue designed for force transmission (Huijing & Baan, 2001).
During development of the muscle, tendon cells attach to the muscle through the
ECM forming the MTU. This formation is a communication between the interac-
tion of the integrins and the ECM molecules secreted by the muscle and tendons, as
well as the contribution of dystroglycan, responsible for muscle binding to the ECM
(Valdivia et al., 2017). The MTU is one of the two structures responsible for the
transmission of a mechanical force between the outside and inside of muscle cells,
based on the structural relationship between the cytoskeleton proteins, talin and
vinculin, and components of the ECM, with the other structure being the costamere
(Bozyczko, Decker, Muschler, & Horwitz, 1989; Pardo et al., 1983). Vinculin and
talin function to connect the actin filaments to the muscle sarcolemma with regard
to the MTU, as well as maintaining the internal myofibrillar structure and the trans-
mission of tension during contraction with regard to the costameres (Bozyczko
et al., 1989).
The fingerlike projections are interdigitations of the sarcolemma, where actin
filaments extending from the last Z-disc are connected to the subsarcolemmal pro-
teins indirectly interacting with the tendinous ECM proteins (Charvet, Ruggiero, &
Le Guellec, 2012). Two separate transmembrane linkage systems are present in the
MTU, each structurally linked by laminin, specifically laminin 211 (Charvet et al.,
2012). Laminin is a multifunctional macromolecule found in the basement mem-
brane and is the most abundant structural non-collagenous glycoprotein in the ECM
(Aumailley & Smyth, 1998). The systems linked by laminin are the dystrophin-
glycoprotein complex and the α7β1 integrin, as discussed above.
Suggestion that the “chain is no stronger than its weakest link” exemplifies the
importance of the relationship of the sarcolemma to the basement membrane, a thin
highly specialised sheet of ECM surrounding muscle. To insure a mechanical rein-
forcement of the sarcolemma during the cycle of contraction and relaxation, it is
necessary that an intact link exists between the intracellular cytoskeleton and the
surrounding basement membrane (Holmberg & Durbeej, 2013). This membrane
consists of two layers, the basal and reticular lamina, with the latter being composed
mainly of collagen and connected to the endomysium (Holmberg & Durbeej, 2013).
The basal lamina, directly linked to the sarcolemma, consists of the lamina lucida
and densa. Since it is beyond the scope of this manuscript to go into detail regarding
laminin, the basement membrane, and the MTU, the reader is directed to material at
the end of the chapter. What is presented is a pathway connecting the muscle to the
MTU and to the tendon, the transmembrane systems, the structures of the ECM, and
the cytoskeletal proteins.
Strongly present in the MTU, the biological functions of the laminin 211 are
dependent on both the dystrophin-glycoprotein complex and the α7β1 integrin link-
age system. As mentioned in the costamere section, the majority of integrins
involved in cell-ECM adhesion share a common β1 subunit, with the main integrin
in adult skeletal muscle being α7β1 (Song, Wang, Foster, Biesler, & Kaufman,
1992). This is localised peripherally around fibres enriched at the MTU, as well as
the neuromuscular junctions (Bao, Lakonishok, Kaufman, & Horwitz, 1993). The
α7 subunit, highly expressed at the MTU, exhibits a weaker presence in other
regions of the plasma membrane, and is also a determinant of junctional specificity,
serving to distinguish the MTU from other adherens junctions (Bao et al., 1993).
The appearance of α7β1 integrin at the MTU correlates with insertion of myofibrils
into subsarcolemmal densities and folding of the junctional membrane, playing a
role in both the formation and integrity of the MTU, as well as force generation
(Bao et al., 1993). The connection between the ECM and the α7β1 integrin is impor-
tant for the transmission of mechanical forces and maintenance of skeletal muscle
fibres (Grounds, Sorokin, & White, 2005).
Of the dystrophin-glycoprotein complex, α- and β-dystroglycans are major non-
integrin cell surface receptors necessary for binding to components of the surround-
ing basement membrane (Holmberg & Durbeej, 2013; Michele & Campbell, 2003).
The extracellular α-dystroglycan protein binds to laminin 211, a component of the
ECM, and to β-dystroglycan, with β-dystroglycan connecting the cytoskeleton to
the ECM by binding to dystrophin, which in turn combines to the actin cytoskele-
ton, completing a pathway responsible for the transfer of a mechanical signal
between the ECM and cytoskeleton (Campbell, 1995).
In summary, the MTU is a highly specialised region between the muscle and
tendon. It forms an integrated mechanical unit with the last sarcomere of the muscle
and its associated intracellular contractile proteins, and the connective tissue pro-
teins of the tendinous-enriched type I collagen molecules of the tendon ECM matrix
(Charvet et al., 2012; De Palma, Marinelli, Pavan, & Bertoni-Freddari, 2011). The
collagen fibrils of the tendon insert into deep recesses formed between the cylindri-
cally folded fingerlike processes of muscle cells increasing the contact area between
the muscle and tendon collagen fibres. This connection, in shear, is similar to the
magnitude and character of a shear stress at focal adhesions. It has been suggested
that since these structures express similar protein compositions, they are analogous,
for cells adhere tightly to and exert force upon their respective ECM structures
(Tidball, O’Halloran, & Burridge, 1986). The cytoskeletal proteins prominently
localised at focal adhesions and the MTU are vinculin, talin, and α-actinin, key
players in cell signalling and adhesion. As discussed above, these proteins are ideal
candidates for mechanotransduction, since the application of force stimulates
recruitment of vinculin to bind to the exposed sites of the unfolded talin protein
(Haining et al., 2016). Vinculin is found in high concentrations at both focal adhe-
sion complexes (Galbraith et al., 2002) and the MTU. The expression of talin at the
MTU is similar to costameres and, likewise, regulated by mechanical loading. At
the MTU, it possesses a specialised function in force coupling, important in main-
taining the MTU under mechanical stress (Valdivia et al., 2017). It is important to
note that the MTU is a dynamic structure where new sarcomeres are added in this
junction in response to stretch-induced hypertrophy (Garrett, Nikolaou, Rtibbeck,
Glisson, & Seaber, 1988; Williams & Goldspink, 1971). Passive stretch, within a
physiological range, results in a 2% strain on the tendon but an 8% strain in the
MTU demonstrating the differences in the viscoelastic properties of the various
regions of the MTU (Magnusson, 1998).
acroscopic and Microscopic Information Processing Levels:

M
Microscopic—Force, Cells, Tissue, and the Extracellular
Matrix (ECM)
Force, Cells, and Tissue
The impact of mechanical forces on tissue has been known for over a century, when
Julius Wolf postulated that bone tissue adapts its structure to the mechanical envi-
ronment (Eyckmans, Boudou, Yu, & Chen, 2011; Ingber, 2004). He observed that
the trabeculae of the bone matched the principal stress lines caused by physical
loading (Eyckmans et al., 2011), such that the coordinated growth of the tissue was
guided by the mechanical forces imparted on it (Orr, Helmke, Blackman, &
Schwartz, 2006). Mechanical force influences and plays a role in the development
and evolution of the tissues (Silver, 2006), with tissues altering their structure to
best meet the mechanical demands placed on them (Mueller & Maluf, 2002). The
adaptive quality of the cell and ultimately the tissue is determined by force, in par-
ticular its response to the temporal, rate, and magnitude characteristics of a mechan-
ical stimulus responsible for activating distinct signalling pathways affecting their
state and function. This ability to adapt suggests that altering and manipulating the
magnitude of the force(s) influences the outcome of the intervention. In addition,
the magnitude, frequency, and duration of force on tissues and cells are dependent
upon the prior loading history (exercise, disuse, overuse) and the composition of the
ECM (tissue type, age, sex, disease, microdamage) (Lavagnino et al., 2015).
Forces interact with biological structures at various and multiple length scales
influencing their forms and functions. In essence, the processing of information in
units of individual molecules is via spatial-, conformational-, force-sensitive-, and
temporal operations such as binding, unbinding, catalysis, degradation, and cross-
linking (Xia & Kanchanawong, 2017). This ability of cells and tissues to sense and
respond to a mechanical force regardless of its origin is central to many aspects of
biology. The common denominator for such a response is cell shape, more precisely
the deviation from “normal” shape, since all cells have a unique morphology (size
and shape). This change in morphology has been associated with changes in cellular
function since the maintenance of homeostasis is a catalyst of adjusting to the
various stresses imparted on the cell. Biomechanical signals sensed by cells in vivo,
are associated with cellular deformation, mechanical stress, as well as deformation
of the nucleus in response to elongation (Freytes, Wan, & Vunjak-Novakovic, 2009).
In addition, any diffusible factors directing cellular function are either secreted by
the cells themselves (autocrine), produced by other cell types (paracrine), or are
Macroscopic and Microscopic Information Processing Levels: Microscopic—Force… 43
transported through the bloodstream (endocrine) (Freytes et al., 2009). With all
biological tissues and their cells, the ECM and molecular factors incorporated into
the ECM are responsible for altering cell function. Residual tensile “prestress” in
the cytoskeleton, tensile strengths, and elastic stiffness (deformability) is influenced
by the ECM, through attachment of the cell to the matrix (matrix ligand-integrin
linkage), the cell-cell connections, and contractility. As suggested above, force(s)
can be applied by the cell’s own cytoskeleton, or from external sources, influencing
the adhesion(s) mediated by the transmembrane integrins responsible for triggering
intracellular signals remodelling the ECM (Schwartz, 2010).
Physical force(s) (i.e. tension, compression, rotation, bend, shear, and gravity)
(Fig. 2.11) place different structural and functional demands on tissues at the cel-
lular level (Ingber, 1997; Vandenburgh, Hatfaludy, Karlisch, & Shansky, 1991).
Force is both static and dynamic in nature (Watson, 1991), with the level of expo-
sure over a given area defined by the magnitude, time, and direction of the stress
application (Mueller & Maluf, 2002). For instance, if a cell protein undergoes a
structural change from an extended to a contracted state, the application of the
stretching force is responsible for changing its energy state (Khan & Sheetz, 1997).
The direct response of the adhesions to the force is responsible for the recruitment
of the ECM proteins.
Fig. 2.11 Forces. (Published with kind permission of copyright © Nikos C. Apostolopoulos 2018.
All rights reserved)
Forces have been increasingly recognised as major regulators of cell structure

and function (Jamney & Mcculloch, 2007; Khan & Sheetz, 1997) modulating
almost all aspects of cell function, including growth, differentiation, migration,
gene expression, protein synthesis, and apoptosis (Alenghat & Ingber, 2002).
Actively contracting and innervated muscle fibres, subjected to active and passive
tensions, can regulate the growth of the myofibre both longitudinally (Williams &
Goldspink, 1971) and cross-sectionally (Goldberg, Etlingger, Goldspink, &
Jablecki, 1975). Mechanical forces affect the form and function of tissues directly,
with the effects of compression on the bone and cartilage and tension on the muscle
being several examples (Alenghat & Ingber, 2002).
Each organism is constantly subjected to either an external mechanical stress
(e.g. gravity, movements) or internal (contractile and hemodynamic) forces gener-
ated by both muscle and non-muscle cells (Chiquet, 1999). In processes ranging
from the contraction of muscles to the alignment of the chromosomes at the meta-
phase plate, forces must be adjusted to appropriate levels by cells in order for them
to function properly (Khan & Sheetz, 1997), suggesting that the ability for muscle
tissue to respond to mechanical force is central to and relevant to the restoration of
its structure and function (Orr et al., 2006).
Since tissue structures are dynamic and change in response to the physical
demands placed on them, their growth and development are direct responses to the
force (Frost, 1990). Passive stretch in young animals has been observed to cause an
increase in muscle tension, stimulating skeletal muscle growth, by inducing an
increase in protein accumulation in the muscle tissue (Goldspink, 1977; Vandenburgh,
Hatfaludy, Sohar, & Shansky, 1990). It has been observed that mechanical tension
in the form of a “passive” stretch is associated with hypertrophy of the skeletal
muscle following a tenotomy of the gastrocnemius muscle (Schiaffino & Hanzlikova,
1970). This mechanical “tension” is one of the basic “trophic” factors acting on
skeletal muscle, exerting a fundamental regulatory function, adapting the muscle to
variable physiological demands (Schiaffino & Hanzlikova, 1970). Therefore, “con-
tinuous integration” of the internal responses to a mechanical force is integral to the
homeostasis of the tissue, a biological adaptation and regulation of the internal
milieu (Schulkin, 2004).
The Extracellular Matrix (ECM)
The relationship of the human body to the environment is not a linear cause-effect
for the body is a highly intermeshed open energetic system. Its response to change,
more importantly its behaviour to change, is informed by non-linear paradigms,
most notably chaos theory and complex adaptive systems, a quantum rather than a
linear event. By referring to the chaos theory, we are stating that the response of the
body is sensitive to initial conditions that are highly variable and often difficult to
predict, since many systems are involved in coherent responses to environmental
cues (i.e. mechanical stress). The complex adaptive system involves multiple
component parts interacting in a non-linear manner. The information processed

from both the micro- and macroenvironments are important to maintain and organ-
ise the structure and function of the body. At the micro level, a large part of the
information is based on a reciprocal relationship between the cells and the ECM,
which was recognised over 30 years ago and is still a central concept in cell biology
(Bissell & Aggeler, 1987).
The ECM in the muscle, is dynamically remodelled, according to the loads imposed
on it during muscle growth, exercise, and as a response to damage (Purslow, 2008). It
is a complex amalgamation of macromolecules (collagens, non-collagenous glyco-
proteins, elastin, and proteoglycans) capable of self-assembly, predominantly via non-
covalent bonds (Kresse & Schonherr, 2001). At one time it was thought of as an inert,
supportive scaffold, stabilising the physical structure of tissue; however, evidence from
cell culture experiments suggests that it is a dynamic and complex environment char-
acterised by biophysical, mechanical, and biochemical properties specific to each tis-
sue (i.e. bone, muscle tendon) (Bowen, Jenkins, & Fraser, 2013; Lee & Nelson, 2012).
The ECM is important in developmental and regenerative processes, transmembrane
signalling, and force transmission (Badylak, 2002), serving as a reservoir for growth
factors and cytokines, modulating their activation and turnover (Kresse & Schonherr,
2001). This dynamic structure adapts to physiological demands placed upon it, chang-
ing and remodelling, decreasing susceptibility to stress, modifying mechanical and
viscoelastic properties, and increasing load resistance (Voermans et al., 2008). A study
examining myoblast proliferation and differentiation on solubilised and 3D decellular-
ised quadriceps muscle matrices of mice, suggests that in vivo muscle ECM is respon-
sible for guiding cell positioning (Charturvedi et al., 2015).
A hierarchical organisation of ECM zones, corresponding to the endo-, peri-, and
epimysium, the basement membrane of skeletal muscle, and the MTU exists, with
evidence suggesting that the perimysium is continuous with the tendon since both
consist of type I collagen (Gillies & Lieber, 2011). Since these zones overlap, clas-
sification of the components of the skeletal muscle consists of several features
defined by histological, ultrastructural, and molecular appearances (Fig. 2.12).
Fig. 2.12 Classification of skeletal muscle ECM. (Published with kind permission of copyright ©
A symbiotic and reciprocal relationship exists between the cells and the ECM of
each area, influenced by the biological relevance of external cues essential for their
function and maintenance. For instance, cells encapsulated in biomimetic hydro-
gels, have been observed to remodel their surrounding matrix, producing new ECM
molecules, with this remodelling behaviour influenced by several external cues such
as the type of hydrogel and the absence or presence of mechanical and biochemical
stimuli (Ahearne, 2014).
To date, three areas of biological relevance exist: the physical properties (stiff-
ness, viscoelasticity, tensile, compressive, shear), the spatial organisation (size,
shape, morphology of adhesion surface), and the biochemical complexity of matrix
molecules (collagens, laminins, fibronectin) (Akhmanova, Osidak, Domogatsky,
Rodin, & Domogatskaya, 2015). These are fundamental to the form and function of
the connective tissue, regulating the behaviour of cells, as well as determining both
the development and maintenance of tissue homeostasis in response to force, both
internally and externally (Mammoto & Ingber, 2010). By either a direct or indirect
action, the ECM provides the structural foundation for mechanical integrity and tis-
sue function, influencing and regulating the availability of growth factors and cyto-
kines (Hynes, 2009; Pickup, Mouw, & Weaver, 2014). Regulating the production,
degradation, and remodelling of its components, the ECM supports tissue develop-
ment, function, and repair (Lu, Takai, Weaver, & Werb, 2011), for modulation of
cell binding to the ECM is what drives cell growth and adaptation. These compo-
nents, their relative amounts and organisation, and the ECM molecular scaffold are
unique for each tissue, reflecting the specific functions required for the cells present
in the tissue (Gattazzo, Urciuolo, & Bonaldo, 2014).
A substantial part of the volume of tissue is extracellular space, with their cells
being joined, supported, and surrounded by an intricate network of macromolecules
forming the ECM (Alberts et al., 2002). All cells at one time or another make con-
tact with the ECM, either continuously or during important phases (Hynes, 2009).
Cells have a unique relationship with their environment, in particular to mechanical
stress, and coupled with the ECM proteins, are responsible for maintaining their
shape, while providing structural support for tissues (Hynes, 2009). The magnitude
of an external force can affect the response of the cell, altering the geometry of the
ECM, for cells within connective tissue establish and maintain the ECM during
development, remodelling it during adaptation, and repairing it in response to injury
(Humphrey, Dufresne, & Schwartz, 2014). In previous sections, the importance of
the communication between the ECM ligand and its specific cell integrin (i.e.
vinculin-talin-integrins, costameres) was presented. This interactive relationship is
responsible for the cell’s direct reaction to an externally applied or internally created
force. The transmission of information to the cell provides the cues to process any
changes to the topography of the surrounding ECM, such as its rigidity, deform-
ability, and its anisotropy (Bershadsky, Balaban, & Geiger, 2003; Chen, 2008;
Geiger & Bershadsky, 2002). This allows the cell to actively modify its microenvi-
ronment by synthesising or degrading the ECM, secreting cytokines, and communi-
cating with other cells (Freytes et al., 2009).
The stiffness (rigidity) or elasticity of the ECM is an important material property

of connective tissue. Stiffness allows cells to sense the external forces, and respond
to the environment in an appropriate manner, via the process of mechanotransduc-
tion (Mammoto & Ingber, 2010). This property is a measure of the relationship
between the applied force and its compliancy (i.e. stretchability), determining its
ability to adapt to stress, as defined by the magnitude and rate of application of an
external or internal stimulus. As previously discussed, cell adhesions formed by
integrin receptors, are key and important transmembrane structures that process
diverse sources of signals, and are the sites of the transmission of forces between the
cytoskeleton and the ECM, with the ECM playing a central role in transducing the
effects of force to regulate cell functions (Ross et al., 2013). These external and
internal inputs influence cell-cell and cell-matrix contact, since any exposure to a
mechanical perturbation is felt with and influenced by the ECM (Discher, Mooney,
& Zandstra, 2009; Geiger, Spatz, & Bershadsky, 2009). In addition to magnitude,
the frequency and duration of the mechanical stimuli on cells are also determined by
their prior loading history (exercise, disuse, overuse) (Tidball & Quan, 1992).
The ECM is more plentiful than the cells it surrounds, composed of various pro-
teins and polysaccharides secreted locally by the cells themselves, and assembled in
an organised meshwork (Sapir & Tzlil, 2017). The hallmark of the ECM, is that it
can be dynamically remodelled, tailored to both the structure and function of each
tissue, reflecting the tissues inherent physiological state (Egebald, Rasch, & Weaver,
2010; Hynes, 2009). The mechanical properties of a tissue are determined by the
ECM composition and orientation, in particular by the mechanical coupling of the
cell matrix (Qi et al., 2006). An example of the importance of this coupling was
provided by an experiment which observed that the elasticity of the matrix was
responsible for specifying the lineage of a cell towards a neuron, a myoblast, or an
osteoblast, by influencing the differentiation of a mesenchymal stem cell into a cell
type matching the elastic properties of the substrate (Engler, Sen, Sweeney, &
Discher, 2006). In most soft tissues (skin, muscle, brain), the ECM and the function
of adherent cells contribute to and establish this elastic microenvironment (Discher
et al., 2005). The ability to adapt to the substrate and its inherent functional require-
ments, is responsible for the diverse forms of tissues, such as becoming calcified to
form the rock-hard structures of the bone or teeth, or adopt the ropelike organisation
that gives tendons their enormous tensile strength (Alberts et al., 2002).
When referring to the relationship between the cells, the ECM, and the mechani-
cal load, what unfolds is a simplified comprehensive summary of key components,
the substrates, the effectors, and the sensors (Humphrey et al., 2014). The ECM is
composed of a number of molecules belonging to three major families of matrix
components, the macromolecules (the substrate), regulated by diverse biochemical
factors, including growth factors, cytokines, and hormones: elastin(s) and fibrillar
collagens, and the proteoglycans (PGs), with related glycosaminoglycans (GAGs)
(Alberts et al., 2002; Bowen et al., 2013; Humphrey et al., 2014; Hynes & Naba,
2012; Lee & Nelson, 2012). The fibroblasts (the effector) are the primary cells
in connective tissue responsible for building, maintaining, and organising the
mechanical homeostasis of the matrix, producing and secreting the matrix

macromolecules (Humphrey et al., 2014). And finally, the integrins (the sensors),
are the main cellular components mediating the sensing and regulation of ECM
mechanics (Humphrey et al., 2014). Presented below and summarised in Fig. 2.13,
are the fundamental and key players responsible for governing the cell-ECM inter-
actions. For a more in-depth information, the reader is directed to further readings
at the end of the chapter.
The Substrate
The elastic fibres of tissues are composed mainly of elastin, a significant structural
component of the ECM. Elastin, a chemically inert insoluble polymer, is one of the
most stable constituents of the ECM of connective tissue, endowing them with
extensibility and resilience, responsible for mechanical memory (ability to recoil
back to their homeostatic state) (Baldwin, Simpson, Steer, Cain, & Kielty, 2013). It
is very important in connective tissue subjected to repetitive distension and physical
stress (i.e. elastic arteries). Expression and the synthesis of elastin typically occurs
in fibroblasts and chondrocytes, with elevated levels of elastin synthesis observed
following injuries, and as a consequence of pathogenic conditions (Rodgers &
Weiss, 2005).
Fibrillar collagens are the most abundant in vertebrates, playing a structural role
by contributing to the shape, molecular architecture, and mechanical properties of
tissue (i.e. resistance to traction in ligaments) (Ricard-Blum, 2011). They are vital
for transmission of force during muscular contraction (Kjaer, 2004). Consisting of
five members synthesised by connective tissue cells, in particular fibroblasts, osteo-
blasts, and chondrocytes, the major and abundant types are collagens I, II, and III,
with the minor and sparse types being collagens V and XI (Ricard-Blum & Ruggiero,
2005). In contrast to elastin, fibrillar collagens bestow stiffness and strength to con-
nective tissues (Humphrey et al., 2014). It has been observed that similar to contrac-
tile proteins, human tendon and muscle collagen are highly responsive to exercise,
Fig. 2.13 Key players in mechanical homeostasis. (Published with kind permission of copyright
due to the sensing and chemical transduction of a mechanical stress (Miller et al.,
2005). This mechanotransduction process via the ECM, that is, the increased syn-
thesis of collagen from the fibroblasts, is dependent upon an interplay between a
mechanical load and the integrin(s) (Chiquet, Renedo, Huber, & Fluck, 2003; Ingber
et al., 1994).
Proteoglycans, well-known components of the ECM, include cartilage, connec-
tive tissue, and cell surface molecules (Selkirk, 2000). These sugar-modified pro-
teins consist of a protein core, with one or more long unbranched sugar polymers
(glycosaminoglycans) attached through the process of glycosylation. Proteoglycans
are responsible for providing a key stabilising force for proteins within microenvi-
ronments (Arey, 2012). Similar to ECM molecules and adhesion receptors, proteo-
glycans are quite diverse, with some being essential for the function and development
of the skeleton, immune, and central nervous systems (Couchman, 2003). The vari-
ous combinations of proteins (matrix, cell surface transmembrane) and glycosami-
noglycan chains [hyaluronan (HA), chondroitin (CS), keratin (KS), dermatan (DS),
and heparan (HS) sulphate] found in vertebrates account for the diverse proteogly-
can molecules (Perrimon & Bernfield, 2001; Yanagishita, 1993). This structural
diversity is responsible for various functions attributed to glycoproteins. For
instance, syndecan, a family of transmembrane proteoglycans via their heparan
chain, binds growth factors, ECM components, protease inhibitors, chemokines,
and enzymes, and plays an active role in signal transduction (Perrimon & Bernfield,
2001). All cellular processes involving interactions at the cell surface (i.e. cell-
matrix, cell-cell, ligand-receptor) involve proteoglycans, for these molecules avidly
bind proteins and are abundant at the cell surface (Yanagishita, 1993).
The Effectors
The fibroblasts, metabolically active cells, are the primary mononuclear cell types
responsible for the formation of the majority of ECM components including, col-
lagen, fibronectin, proteoglycans, tenascin, and laminin (Gillies & Lieber, 2011;
McNulty, 2006). Although skeletal muscle myoblasts are associated with produc-
tion of collagens, fibroblasts are crucial for the assembly of collagen into a func-
tional ECM (Chapman, Meza, & Lieber, 2016). Lying within the interstitial space
between muscle fibres, fibroblasts are responsible for the production of the majority
of skeletal muscle ECM (Gillies & Lieber, 2011). Coordinating the macromole-
cules, they regulate the synthetic and mechanical machinery, providing the overall
structural organisation and mechanical properties of the tissue (Humphrey et al.,
2014). With fibroblasts continually synthesising ECM proteins, a major function is
the production and the homeostatic maintenance of the ECM. The skeletal muscle
fibroblasts secrete and assemble fibrillar collagen isoforms composed primarily of
collagens I and III (Light & Champion, 1984). These fibrillar molecules bear tre-
mendous stress within the muscle, providing the ECM with the ability to transmit
both longitudinal and lateral force from the muscle to tendons (Chapman et al.,
2016; Kjaer, 2004; Tidball, 1991).
The Sensors
The integrins, mentioned above, are the main cellular components mediating the
sensing and regulation of the ECM mechanics (Humphrey et al., 2014). They are
essential for binding matrix proteins, the associated cytoskeletal and signalling
proteins of focal adhesions, and the actomyosin cytoskeleton (Humphrey et al.,
2014). Focal adhesions, are clusters of integrin transmembrane receptors, coupling
the ECM to the actin cytoskeleton via actin-binding proteins, with sensing, signal-
ling, and force transmission being informed by the actomyosin-integrin-actin-
binding protein-ECM pathway (Ciobanasu, Faivre, & Le Clainche, 2013). The
relationship between the actin-binding proteins, talin and vinculin was discussed
earlier with regard to their importance for force transmission. In particular with
vinculin, its association with focal adhesions, is correlated with the force applied
at the site, with its recruitment to cells being abolished in the absence of talin
(Ciobanasu et al., 2013).
Conclusion
Tissues and cells are exposed to diverse types of extrinsic mechanical forces includ-
ing mechanical stretch (tension), compression, and shear stress (Carver & Goldsmith,
2013). Their behaviour is largely determined and influenced by interactions with
neighbouring cells, systemic mechanical cues, and the skeletal muscle ECM. The
structural integration between the individual muscle fibres and the ECM, with its
collagen content linking tissues together, determines force transmission and absorp-
tion of loading energy, tissue structure maintenance, and repair for tendons, liga-
ments, bone, and muscle (Alexander, 1991; Alexander & Bennett-Clark, 1977;
Gillies & Lieber, 2011; Kjaer, 2004). The altered expression of specific ECM pro-
teins is part of the adaptive response to various types of mechanical stress (Chiquet,
1999). For instance, during diseased or injured states, the cell and the ECM adapt by
altering muscle function accordingly (Gillies & Lieber, 2011). The mechanical
stress caused by these states regulates production of ECM proteins directly, with the
triggering of an intracellular signalling pathway activating the gene, or indirectly,
with release of a paracrine growth factor (Chiquet et al., 2003). It is interesting to
note that the environment surrounding the cell, determines its pattern of gene
expression and phenotype, despite the genome of the cell remaining the same
(Nelson & Bissell, 2006).
An in vitro study observed tension as a requirement for maintaining the pheno-
type typical of tendon fibroblasts, for which collagen I amounts to more than 20%
of their total protein synthesis (Chiquet, 1999; Quinones, Neblock, & Berg, 1986).
Interestingly, the same cells (collagen I) at sites of compression “transdifferentiate”
into fibrocartilage (Benjamin & Ralphs, 1998; Chiquet, 1999), suggesting that the
type of mechanical stress is a basic requirement for proper function. In this manu-
script, the concern is with tension and tensile strength (i.e. resistance to pull) of the
The Strain Factors of Stretching: Intensity, Duration, and Frequency 51
muscle and tendon, in the form of stretching. The tensile strength of the tissue,
specifically the matrix, is based on intra- and intermolecular cross-links and the
orientation and length of the collagen fibrils and fibres (Kjaer, 2004). The connec-
tive tissue of skeletal muscle and tendon is a “plastic” structure with a dynamic
protein turnover possessing the capacity to adapt to changes in the external environ-
ment, such as mechanical loading or inactivity and disuse (Kjaer, 2004). The reali-
sation that mechanical stimuli (load, force) contribute to the health and pathogenesis
of the tissue, illustrates the need to research the magnitude and rate of stretch-
ing that best contributes to the overall health and recovery of the tissue(s) of the
musculoskeletal system: the muscles, the tendons, and the MTU.
he Strain Factors of Stretching: Intensity, Duration,

T
and Frequency
Stretching initiates a stress/strain response on the connective tissue dependent on its

magnitude (intensity) and rate, which is influenced by the biochemical complexity
of the matrix molecules (i.e. collagen, elastin, PGs, and GAGs) (Ransone, Geissler,
Wilson, & Adams, 2012). As mentioned above, these fibrillar molecules bear tre-
mendous stress within the muscle, providing the ECM with the ability to transmit
both longitudinal and lateral force from the muscle to tendons, in response to a time-
varying mechanical stimuli (Chapman et al., 2016; Hoffman, Grashoff, & Schwartz,
2011; Kjaer, 2004; Tidball, 1991); This stress/strain response is responsible for cel-
lular changes as observed in an in vitro study, where the concentration in tenascin C
and collagen XII, two ECM components of fibroblasts, increased by an eightfold
during stretching (Chiquet, 1999).
According to DeDeyne (2001), the mechanical stimulus of stretching influences
the ECM, detected by and transmitted via the integrin into the cell interior activating
a series of nuclear proteins which modify gene transcription and regulate sarco-
meregenesis. Sarcomeregenesis is the synthesis of contractile proteins produced
by specific muscle genes by mechanotransduction (Martins et al., 2013).
Mechanotransduction, the process by which mechanical energy is converted into
biochemical signals, is critical for mediating adaptations to mechanical load in con-
nective tissues which precipitates changes in intracellular biochemistry and gene
expression (Hamill, 1997; Ingber, 2008b; Ko & Mcculloch, 2001). More on mecha-
notransduction will be presented below.
With cellular response to mechanical force coupled to the organisation of the
ECM (Chen, Tan, & Tien, 2004), the ECM, similar to hormones and growth factors,
plays an important role in cell growth and behaviour (Daniels & Solursh, 1991;
Shimizu & Shaw, 1991). As discussed above, this interaction between the ECM and
cell is largely mediated by the integrins, bridging the cell-ECM interactions (Hynes,
1992; Meredith, Fazeli, & Schwartz, 1993). The adhesion facilitated by the integ-
rins ensures a mechanical connection between the ECM fibrils with the intracellular
actin cytoskeleton, facilitating the response and adaptation to mechanical stimuli
from the environment (Schwartz, 2010). This adhesion is not only essential for
binding matrix proteins and the associated cytoskeletal and signalling proteins
(Humphrey et al., 2014)., but provides the understanding of how the force generated
by stretching is transmitted to the cytoskeleton.
Regardless of the stretching methods used to facilitate increases in ROM about a
joint, what is common amongst all is the parameters of training: intensity, duration,
and frequency (Marschall, 1999; Mujika et al., 1995). Prescriptions of intensity
(how hard the stretch is, referencing the elongation of the muscle for a given rate of
stretch), duration (time spent), and frequency (how often), are responsible for the
adaptive response of the tissue to stretching or training (Mujika et al., 1995; Seiler,
2010; Young, Elias, & Power, 2006). In addition, the position assumed during
stretching may influence directly or indirectly the intensity of the stretch, for the
force generated prior to and/or during the stretch (passive or active), potentially
alters the response of the muscle and tendon tissue and their components (i.e. col-
lagen) (Apostolopoulos et al., 2015), which respond to altered levels of activity
(Kjaer, 2004). A study comparing a standing to a supine hamstring stretch observed
that the latter isolated the hamstring muscle better, was more comfortable, but more
importantly facilitated a better relaxation response during stretching (Abdel-aziem
et al., 2013).
Although duration and frequency are important in stretching programmes, with
bouts of stretching (30–60 s) improving ROM and flexibility in the human muscle
(Bandy et al., 1997), the intensity, the magnitude of force applied during stretching
(Jacobs & Sciacia, 2011), and the rate of force may be of a greater significance. Too
much force during stretching is associated with an inflammatory response and fibro-
sis (Brand, 1984; Mcclure, Blackburn, & Dusold, 1994). However, unlike duration
and frequency, which are easier to quantify, with numerous articles referring to
them in relation to stretching (Bradley, Olsen, & Portas, 2007; Brandenburg, 2006;
Cornwell, Nelson, & Sidaway, 2002; Feland, Myrer, & Merrill, 2001; Kokkonen,
Nelson, & Cornwell, 1998), intensity is more difficult. In relation to stretching,
intensity is defined as a feeling, a perception associated with a certain amount of
exertion. This relationship between perceived intensity and stimulus strength is a
psychosocial response, an internal representation associated with the magnitude of
the stimulus, evoking a feedback, essentially a relative measure exacting a con-
scious interpretation within the mind (MacKay, 1963). According to psychophysi-
cal laws, this perception or feeling represents a concept referred to as a “magnitude
production” (Stevens, 1971). In other words, the value associated with the intensity
of the stretch conveyed by the individual, is an adjustment in their response,
producing a match to the stimulus. In short, intensity is a qualitative response unique
to individuals. This qualitative nature explains why intensity, with respect to stretch-
ing, remains under researched, accounting for the lack of relevant articles.
Therefore, the aim of the current project is to look at stretch intensity and its
influence on the musculoskeletal tissues, with a particular focus placed on the rela-
tionship of the magnitude of the force generated during stretching and how this may
cause inflammation and the inflammatory response. Stretching imparts a mechani-
cal energy on the body, with stretching intensity (i.e. low vs. high) potentially
Inflammation 53
influencing the mechanical equilibrium between the tension-generating muscle and

tension-resisting tendons. In short, the manipulation of stretch intensity may pro-
vide answers for the proper recovery of the musculoskeletal tissue, thereby improv-
ing its function and athletic performance, as well as for the treatment of various
musculoskeletal disorders, by influencing inflammation and the inflammatory
response.
Inflammation
Inflammation is an essential immune response that enables survival during infection or

injury and maintains tissue homeostasis under a variety of noxious conditions. Inflammation
comes at the cost of a transient decline in tissue function, which can in turn contribute to the
pathogenesis of diseases of altered homeostasis.
… Medzhitov (2010)
Introduction
The history of Inflammation and its description can be traced back to both ancient
Egypt and Greece (Granger & Senchenkova, 2010). Hippocrates, the father of medi-
cine, coined the term oedema to describe inflammation, referring to it as a necessary
process of healing following tissue damage (Granger & Senchenkova, 2010). Based
on a visual observation, the Roman medical writer, Celsus, is credited with describ-
ing the four cardinal signs of acute inflammation: rubor et tumor cum calore et
dolore (redness and swelling, with heat and pain) (Marmelzat, 1977; Medzhitov,
2010). The fifth sign, functio laesa (loss of function), was added a century and a half
later, by the Greco-Roman physician/physiologist, Galen, being a characteristic
sign for both acute and chronic inflammation (Lawrence, Willoughby, & Gilroy,
2002; Rather, 1971; West, 2014). In the eighteenth century, the Scottish surgeon
John Hunter wrote, “inflammation in itself is not to be considered as a disease, but
as a salutary operation consequent to some violence or some disease” (Palmer,
1835), further reinforcing the importance of inflammation with regard to homeosta-
sis of the body.
Inflammation is an important and useful host defence mechanism in response to
a foreign body challenge or tissue injury. It is a necessary information process of our
tissues and their ability to function and survive; however, it can also cause further
damage (Slauson & Cooper, 2002). In 1972, Thomas Lewis wrote, “Our arsenal for
fighting off bacteria is so powerful, and involves so many different defense mecha-
nisms, that we are more in danger from them than the invaders. We live in the midst
of explosive devices; we are mined” (Thomas, 1972).
Inflammation attempts to restore the damaged tissue to its pre-injury state. It
consists of an innate system of cellular and humoral responses (Rock & Kono,
2008; Ward, 2010). It is either acute or chronic, with acute defining a series of
responses beginning within hours and lasting for several days, characterised by the
influx of neutrophils and macrophages, cells of the immune response (Ward, 2010).
The adaptive value of the acute inflammatory response is removal of cellular debris,
necrotic tissue, and the repair of damaged blood vessels, myofibres, and the ECM
(Cannon & St. Pierre, 1998). However, if the injury is persistent or a foreign mate-
rial in the wound cannot be eliminated, this progresses to chronic inflammation,
with a preponderance of lymphocytes and macrophages (Lucke et al., 2015), sug-
gesting that chronic inflammation is not defined by the duration of the response
(Ward, 2010). In order for tissue to return to a normal state, what is needed is a
moderation of the four components of a typical inflammatory response: the induc-
ing stimulus, the sensors, the inflammatory mediators, and the target tissues
(Medzhitov, 2010). The inducing stimulus (i.e. muscle or tissue damage, injury,
infections, etc.) initiates the inflammatory response, with the outcome (i.e. apop-
totic or damaged cells) being detected by the sensors of the inflammatory pathway
(i.e. resident macrophages, toll-like receptors, etc.) at the site of damage. These
sensors induce production of the inflammatory mediators [i.e. cytokines (IL-1β,
IL-6, and TNF-α)], which act locally (site of damage) and systemically (other parts
of the body), effecting various target tissues (i.e. blood vessels, endothelial cells,
liver, etc.) of the inflammatory response (Haslett, 1992; Medzhitov, 2010).
The disturbance of the microcirculation (i.e. infection, tissue injury) begins the
inflammatory response, with leukocytes and serum proteins moving from the blood
to the extravascular tissue (Lawrence et al., 2002; Medhitov, 2008). This is con-
trolled and regulated by the release of vasoactive and chemotactic mediators
(Lawrence et al., 2002). The nature of the inflammatory trigger (i.e. infection, tissue
damage) is responsible for the path preceding the inflammatory response (i.e. bacte-
rial, viral infections, tissue damage, etc.) (Medzhitov, 2010). With this present work,
the concern is the response to a muscle disturbance caused by a mechanical stimulus
(i.e. stretching), since this occurs in the absence of an infection.
Several mechanisms are responsible for inflammation: the injurious stimulation
of mast cells and nerves, bleeding caused by tissue damage, cell death due to injury,
contusions, and eccentric exercise (Rock & Kono, 2008; Smith, 1991, 1994).
Mechanical injury to the myofibre activates an immediate necrosis along the entire
length of the myofibre exposing the sarcoplasm (Li, Cummins, & Huard, 2001). The
myofibres nearest the damage undergo a hypercontraction, tearing apart into a series
of irregular dense masses of myofilaments in the form of contraction clots (Warren,
Lowe, & Armstrong, 1993). These clots activate an extracellular protein kinase
responsible for initiating degeneration of muscle tissue and local inflammation
(Aronson et al., 1998).
Eccentric exercise and acute stretch injuries are examples of mechanical stimuli,
given that the force generated causes an excessive overload of the contractile ele-
ments of the skeletal muscle exceeding its habitual requirements (Toumi & Best,
2003). Structurally, there is a disarrangement of the myofilament in the sarcomeres,
damage to the sarcolemma, loss of fibre integrity, and the subsequent leakage of the
muscle proteins into the blood (Jones, Newham, Round, & Tolfree, 1986). This
functional change results in a decrease or loss in muscle force, changes in mechanical
Inflammation 55
(Brockett, Morgan, & Proske, 2001) as well as proprioceptive properties (Walsh,

Allen, Gandevia, & Proske, 2006), and is responsible for triggering an acute
response (Hellsten, Frandsen, Orthenblad, & Sjodin, 1997). According to Tidball
(2005), the extent of the inflammatory response is determined by the degree of
muscle damage, the magnitude of the inflammation, and the injury-specific interac-
tion between the invading inflammatory cells and the damaged muscle. Muscle
inflammation is defined by several phases, destruction, repair, and remodelling
(Jarvinen, Jarvinen, Kaariainen, Kalimo, & Jarvinen, 2005), with each characterised
by the appearance of predominant inflammatory cell types (Philippou et al., 2012).
eutrophils, Macrophages, Cytokines, and Acute Phase

N
Response
Neutrophils and Macrophages
The release of reactive oxygen species by necrotic cells caused by damage to mus-
cle tissue triggers the inflammatory response (inducing stimulus) (Cheung, Hume,
& Maxwell, 2003). Once within the interstitium, the inflammatory cells activate
the resident satellite cells (the sensors) releasing a chemotactic agent, resulting in
the infiltration of circulating inflammatory cells (Clarkson & Hubal, 2002; Robertson
et al., 1993). These inflammatory cells migrate into the site of inflammation through
the endothelial wall of the skeletal muscle cells, via the process of rolling, adhesion,
and transmigration (Ley, Laudanna, Cybulsky, & Noursharhg, 2007).
In the early stages of acute inflammation, the first responders are the neutrophils
and polymorphonuclear leukocytes, recruited to the inflammatory site. Their activa-
tion increases their longevity several fold, which ensures their presence enabling
them to carry out complex activities contributing to the resolution of inflammation
or shaping the adaptive immune response (Kolaczkowska & Kubes, 2013).
Neutrophils are detected within the first hour, peaking between 24 and 48 h and
remaining for up to 5 days (Smith, Kruger, Smith, & Myburgh, 2008; Tidball, 2005).
In addition to their phagocytic activity, they release proteases aiding in the degrada-
tion and removal of the cellular debris (Tidball, 2005). Neutrophils are gradually
replaced by macrophages, appearing approximately 2 days post damage, further
contributing to the degradation of the damaged tissue and the removal of necrotic
cellular debris (Lawrence et al., 2002; Philippou et al., 2012). In addition, macro-
phages play a key role in repair (Cannon & St. Pierre, 1998; McLennan, 1993).
Unlike neutrophils, macrophages consist of two phenotypes, the phagocytic pro-
inflammatory, responsible for removal of cellular debris and necrotic tissue (neutro-
phils), and the non-phagocytic anti-inflammatory, involved in muscle repair, which
peaks 4 days post damage and remains elevated for many days afterwards (Malm
et al., 2000; Smith et al., 2008; Tidball, 2005; Tidball & Villalta, 2010). Both neu-
trophils and macrophages are responsible for the release of cytokines. More will be
discussed below.
Cytokines
Cytokines, are small glycoproteins released by activated immune cells regulating

inflammation. They are produced by a number of cell types (i.e. leukocytes) (Khan,
2008). In response to tissue damage and infection, cytokines function as intercel-
lular messengers responsible for the movement of immune cells towards the site of
inflammation (Kushner, 1993; Zhang & An, 2007). These soluble mediators are
multifunctional, such that more than one cytokine is capable of acting on the same
target cell negotiating similar functions (Akira, Hirano, Taga, & Kishimoto, 1990).
The term cytokine is a general term with specific terms referring to their origin.
Cytokines are subdivided into either pro- or anti-inflammatory. They act locally in
an autocrine (a hormone action binding to receptors on and affecting the function of
the same cell that produced it) (The Free Dictionary, 2016), or a paracrine (a com-
munication between cells producing a signal-inducing changes to nearby cells)
manner, and are capable of an endocrine communication, responsible for cell-to-
cell messaging over a long distance affecting a target organ (i.e. liver). Cytokines
are characterised by pleiotropy (multiple biological actions), redundancy (shared
biological actions), synergy (two or more cytokines’ additive effect), and antago-
nism (two or more cytokines’ inhibitory effects), with these characteristics respon-
sible for creating a complex and intricate network (Nicola, 1994; Veskler, 2005).
Although muscle damage stimulates a local cytokine cascade, initiated by various
cell types (fibroblasts, neutrophils, and macrophages), a clear-cut and specific con-
tribution of each cell type and the subsequent specific roles of each cytokine is very
difficult to determine (Smith et al., 2008). Types of molecules included under the
cytokine umbrella are the tumour necrosis factors (TNF-x), the growth factors
(x-GF), and the interleukins (IL-x) (secreted by some leukocytes acting on other
leukocytes) (Fig. 2.14).
Neutrophils, the first responders of the immune system migrate to the affected
sites in response to injury (Kim & Luster, 2015). They are a source of chemokines
produced in the bone marrow (Furze & Rankin, 2008), being a group of cytokines
specifically released to recruit discrete leukocyte subpopulations (Scapini et al.,
2000; Von Vietinghoff & Ley, 2008). One such chemokine is the pro-inflammatory
cytokine IL-1β, a potent chemoattractant (a chemical agent inducing a cell to
migrate towards it) for macrophages (Smith et al., 2008), with the neutrophils and
macrophages responsible for the release of another cytokine, TNF-α. Both IL-1β
and TNF-α, besides initiating the degradation of the damaged muscle tissue (Peake,
Nosaka, & Suzuki, 2005), are responsible for the expression of a third pro-
inflammatory cytokine, IL-6 (Frost & Lang, 2005). IL-6, a known pleiotropic
(affects the activity of multiple cell types) cytokine is released into circulation in
response to multiple homeostatic perturbations including injury, trauma, and acute
infections (Streetz, Wustefeld, Klein, Manns, & Trautwein, 2001). These three cyto-
kines form a complex intricate network with the expression of each influencing the
other, with IL-1 and TNF-α being potent inducers of IL-6, and IL-6 regulating the
expression of TNF-α (Akira et al., 1990; Akira, Taga, & Kishimoto, 1993; Mcgee,
Bamberg, Vitukus, & Mcghee, 1995). This synergistic relationship is an attempt to
Inflammation 57
Fig. 2.14 Summary of cytokines. (Published with kind permission of copyright © Nikos
regulate the immune response and the inflammatory reaction (Akira et al., 1990,
1993; Streetz et al., 2001). In addition, IL-6 production in contrast to IL-1β and
TNF-α has been linked to a contraction-induced response by skeletal muscle (Smith
et al., 2008). The release of these cytokines locally is responsible for initiating a
systemic response to tissue damage, the acute phase response (Streetz et al., 2001).
Acute Phase Response
The acute phase response is activated as a feedback to disturbances of the homeo-

stasis of the organism (Kushner, 1982) (Fig. 2.15). It is linked to both a major adap-
tive and defensive role regarding tissue injury and infection. The acute phase
response, an intrinsic response, is initiated after the first few days following the
inducing stimulus and is associated with a vast number of changes, both systemic
and metabolic (Kushner, 1982). Its objective is the containment and destruction of
infectious agents, removal of damaged tissue, and assisting in repair (Kushner,
1982). As mentioned above, IL-1β, TNF-α, and IL-6 are responsible for its activa-
tion; however, although IL-1 and TNF-α are considered mediators with regard to
initiating the acute phase response, they are not responsible for inducing a full acute
phase response (Akira et al., 1990); that role primarily belongs to IL-6, initially
referred to as a hepatocyte-stimulating factor (Fuller & Grenett, 1989; Gruys,
Toussaint, Niewold, & Koopmans, 2005; Ramadori, Van Damme, Rieder, & Meyer
Zum Buschenfelde, 1988; Streetz et al., 2001) (Fig. 2.5).
Fig. 2.15 Acute phase response. (Published with kind permission of copyright © Nikos
The acute phase response refers to changes in the concentrations of numerous

acute phase proteins produced in the liver (Kushner, 1993; Streetz et al., 2001). It is
primarily induced by IL-6, a messenger between the hepatocytes synthesising the
acute phase proteins and the local site of damage (Baumann & Gauldie, 1990;
Whicher & Westacott, 1992). The acute phase proteins are influential in one or more
stages of inflammation, healing, or an adaptation to a noxious stimulus (Gabay &
Kushner, 1999). In humans, C-reactive protein, fibrinogen, and serum amyloid
alpha are acute phase proteins (Jain, Gautam, & Naseem, 2011). Normally present
in trace amounts in the plasma, an averse stimulus initiates a dramatic increase in
their rate of synthesis and concentration by the liver (Kushner, Ganapathi, & Schultz,
1989).
Interest in the present work is with C-reactive protein, in particular high-
sensitivity C-reactive protein (hsCRP), as a measure of inflammation in response to
various stretching intensities. C-reactive protein is present at the very onset of any
infection or tissue injury (Castell et al., 1990; DuClos, 2000; Heinrich, Castell, &
Andus, 1990). With its synthesis not specific to any disease, increases or decreases
in its concentration are proportional to the inflammatory stimulus (Libby, Ridker, &
Maseri, 2002). The expression of C-reactive protein is considered a more accurate
reflection of the acute phase response compared to any other biomarkers (Kilicarslan,
Uysal, & Roach, 2013; Libby et al., 2002). It has a long biological half-life of
19–20 h (Marino & Giotta, 2008; Pepys & Hirschfield, 2003; Vigushin, Pepys, &
Hawkins, 1993). This allows for the stability of concentration levels that are not
subject to a circadian variation such as IL-6 (Meier-Ewert et al., 2001), consisting
Inflammation 59
of a diurnal variation, being low in the morning with higher concentrations mea-
sured both in vivo or ex vivo before bedtime (Meier-Ewert et al., 2001; Sothern
et al., 1995; Vgontzas et al., 1999).
C-reactive protein, which represents the downstream integration of overall cyto-
kine activation, is credited with playing contradictory roles (Libby et al., 2002). The
synthesis of pro-inflammatory cytokines (IL-1α and β, TNF, and IL-6) by C-reactive
protein suggests a magnification of the inflammatory response; however, its synthe-
sis of interleukin-1 receptor antagonist (IL-1ra), in circulating monocytes, suggests
an anti-inflammatory role, with the subsequent suppression of pro-inflammatory
cytokines in tissue macrophages in the presence of IL-6 (Tilg, Trehu, Atkins,
Dinaello, & Meir, 1994). This anti-inflammatory role is partially explained by its
relationship with IL-6 (Samols, Agrawal, & Kushner, 2002), since IL-6 is respon-
sible for synthesis of IL-1ra and the TNF-a antagonists, directly suppressing synthe-
sis of the pro-inflammatory cytokines (IL-1β and TNFα) (Tilg et al., 1994).
Interestingly, with chronic inflammation, IL-6 expresses a pro-inflammatory role
(Srirangan & Choy, 2010), contradicting its beneficial protective role in resolving
acute inflammation (Gabay, 2006). In this capacity, IL-6 favours the accumulation
of mononuclear cells through the continuous secretion of monocyte chemoattrac-
tant protein-1 and anti-apoptotic functions on T-cells at the site of injury (Atreya
et al., 2000). According to Gabay, the expressed synthesis and increased concentra-
tion of IL-6 serves as a transition from acute to chronic inflammation (Gabay, 2006).
Therefore, IL-6 is a pivotal keystone cytokine, for if tissue damage is not resolved
during the acute inflammatory response, its role shifts to perpetuating inflammation
as seen in a condition such as rheumatoid arthritis (Hashizume & Mihara, 2011;
Metsios, Stavropoulos-Kalinoglou, & Kitas, 2015; Srirangan & Choy, 2010; Yoshida
& Tanaka, 2014).
Inflammation and Exercise
In the literature, inflammation and the inflammatory response have been examined
relative to either non-damaging (e.g. concentric) or damaging (e.g. eccentric) exer-
cise, with the study by Canon et al. being the first to suggest that cytokines are
released in response to exercise (Cannon & Kluger, 1983). As stretching is consid-
ered a form of physical activity used by athletes and rehabilitation patients (Page,
2012; Weerapong et al., 2004), by investigating physical activity and its relationship
to the inflammatory response, this provides a plausible explanation of the relation-
ship of stretching to inflammation. Similar to exercise (non-damaging and damag-
ing), efficacy of stretching is determined by the parameters of training: intensity,
duration, and frequency (Marschall, 1999; Mujika et al., 1995; Seiler, 2010). In this
section, both non-damaging and damaging exercise relevant to the inflammatory
response(s) will be discussed.
Non-damaging Exercise and the Inflammatory Response
Since the study by Cannon and Kluger (1983), exercise has been observed to induce
the cytokine cascade characterised by the release of the following specific cyto-
kines, the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) (Drenth et al.,
1995; Sprenger et al., 1992), and the anti-inflammatory cytokines [IL-1ra, TNF-α
antagonists, and IL-10 (cytokine inhibitors)] (Drenth et al., 1995; Ostrowski, Rohde,
Asp, Schjerling, & Pedersen, 1999; Pedersen & Hoffman-Goetz, 2000). With habit-
ual (non-damaging) exercise, a marked increase in the concentration of plasma IL-6
compared to other cytokines occurs (Pedersen & Hoffman-Goetz, 2000), contra-
dicting a study that suggests this increase is due to muscle damage (Bruunsgaard
et al., 1997). Support for the former study was provided by an investigation that
observed the return of IL-6 to resting level postexercise, despite the continued
expression of the two markers for muscle damage, namely, creatine kinase and
myoglobin (Peake et al., 2005). With the immune response dependent on the ability
of leukocytes to transmigrate from circulation to the site of inflammation through
the endothelium, increases in the concentration of adhesion molecules on the sur-
face of leukocytes and endothelial cells occur in response to muscle damage
(Akimoto et al., 2002; Alberts et al., 2002; Carlos & Harlan, 1994; Chen et al.,
2006; Kaplanski, Marin, Montenero-Julian, Mantovani, & Farnarier, 2003;
Reihmane & Dela, 2013; Springer, 1994; Vestweber, 2007). However, during habit-
ual exercise, this increase in adhesion molecules does not occur, remaining rela-
tively unchanged postexercise or during recovery (Chaar et al., 2011; Smith et al.,
2000). Considering that numerous cells from a variety of tissues can produce pro-
inflammatory cytokines (i.e. IL-6, TNF-α), alteration in the concentration levels of
these cytokines is not necessarily reflective of changes in their production by circu-
lating leukocytes (Reihmane & Dela, 2013; Starkie, Rolland, Angus, Anderson, &
Febraio, 2001). Therefore, since the rise in the concentration of plasma IL-6 during
habitual exercise is not due to the circulating leukocytes, this rise is believed to be
associated with muscle contraction (Steensberg et al., 2000, 2001, 2002).
The release and rise of IL-6 from skeletal muscle during exercise (eccentric and
concentric) (Hiscock, Chan, Bisucci, Darby, & Febbraio, 2004) was measured dur-
ing sustained marathon running (Ostrowski, Rohde, Zacho, Asp, & Pedersen, 1998),
the contraction of knee extensor muscles of a single leg during a 5h concentric
exercise (Steensberg et al., 2000), and during cycling (Ullum et al., 1994). With no
changes observed for pre-mRNA IL-6, IL-1α, IL-1β, and TNF-α in the blood mono-
nuclear cells due to habitual exercise, increases in plasma IL-6 was less likely
related to the increased production of cytokines (Ullum et al., 1994). Unlike eccen-
tric exercise, which is associated with micro-injuries to both the muscle and connec-
tive tissue, concentric exercise provides an opportunity to observe the expression of
IL-6 during non-damaging exercise (Macintyre, Reid, & Mckenzie, 1995). The
amount of plasma IL-6 released by the knee extensor muscles was ~100-fold greater
compared to pre-exercise, with levels remaining relatively unchanged in the non-
exercised leg (Steensberg et al., 2000). This several fold rise in the concentration
of IL-6 has been detected in clinical studies concerned with serious infection
Inflammation 61
(Bruunsgaard, Skinhoj, Qvist, & Pedersen, 1999; Damas et al., 1992; Hack et al.,
1989) and observed in a study investigating prolonged running (i.e. a marathon
competition) (Starkie et al., 2001). Unlike the aforementioned studies which had
participants perform a concentric exercise (i.e. bicycling, knee extension exercise)
(Steensberg et al., 2000; Ullum et al., 1994), this study employed an eccentric exer-
cise in the form of running (Starkie et al., 2001). Interestingly, the concentration of
IL-6 associated with the eccentric exercise was not much greater than the concentric
exercise, demonstrating that damage was not a requisite for its release during habit-
ual exercise (Pedersen & Febbraio, 2008) (Fig. 2.16).
An investigation in which the calf muscles of male Wistar rats were electrically
stimulated concentrically or eccentrically, observed a local production of IL-6 in the
exercising leg, with levels for the non-stimulated remaining unchanged (Jonsdottir
et al., 2000), supporting the view that expression of IL-6 is closely related to muscle
contraction. In addition, significant differences in muscle fibre type were not
observed with similar levels of IL-6 measured in both the red and white fibres of the
soleus and gastrocnemius and the gastrocnemius, respectively (Jonsdottir et al.,
2000). Analogous to the studies by Ullum et al. (1994) and Steensberg et al. (2000),
this study confirmed that muscle damage was not a requisite for increases in plasma
IL-6 during habitual exercise, but released in response to muscle contraction
(Jonsdottir et al., 2000).
Fig. 2.16 Relationship of IL-6 relative to concentric and eccentric non-damaging exercise.
(Modified from Pedersen and Febbraio 2008)
Myokines
Myokines are cytokines produced, released, and expressed by muscle fibres exhibit-
ing a paracrine or endocrine effect (Pedersen, Akerstrom, Nielsen, & Fischer, 2007).
They account for exercise-associated immune changes, playing a role in resolving
both exercise-associated metabolic changes following training adaptation (Pedersen
et al., 2007). The myokines in response to habitual exercise are responsible for
expressing IL-6, IL-1ra, IL-8, IL-10, IL-15, and TNF-α into circulation, with TNF-α
appearing in response to very intense long duration exercise (e.g. marathon running)
(Febbraio & Pedersen, 2002, 2005; Nielsen et al., 2007; Starkie et al., 2001; Ullum
et al., 1994). This late release of TNF-α does not precede the expression of IL-6 as
occurs with sepsis or muscle-damaging exercise (Pedersen & Febbraio, 2008)
(Fig. 2.17).
Interleukin-6, IL-8, and IL-15 are classified as myokines since their expression
is regulated by the type of muscular contraction (Marino, Scuderi, Provenzano, &
Bartoccioni, 2011; Pedersen & Febbraio, 2008). Both IL-6 and IL-8 are regulated
by concentric contraction at the protein and mRNA level (Akerstrom et al., 2005;
Chan, Carey, Watt, & Febbraio, 2004; Febbraio & Pedersen, 2002, 2005; Pedersen
et al., 2007), with IL-15 regulated by resistance training (Nielsen et al., 2007).
Production of IL-6 in response to muscle activity has been linked to muscle repair
(Steensberg et al., 2000), with an increased expression observed by regenerating
mice myofibres exposed to a crush trauma (Kurek, Nouri, Kannourakis, Murphy, &
Austin, 1996). Interleukin-8, a chemoattractant cytokine, is produced by a variety of
blood and tissue cells, with a distinct specificity for neutrophils, attracting and acti-
vating them at the site of inflammation and regulating the neutrophil-endothelial
interaction (Bickel, 1993). Interleukin-15, a pleiotropic cytokine expressed at the
mRNA level of numerous normal human tissues (i.e. activated monocytes, dendritic
cells, fibroblasts, and osteoclasts), is involved in both adaptive and innate immune
responses inducing the synthesis of TNF-α (Itsumi, Yoshikai, & Yamada, 2009;
Liew & Mcinnes, 2002).
Fig. 2.17 Comparison of muscle damage (a) vs. habitual exercise (b) induced cytokines (note:
during muscle damage IL-6 release is preceded by TNF-α). (Modified from Pedersen and Febbraio
2008)
Inflammation 63
In order to determine the source of IL-6 in contracting muscle, the reverse

transcription-polymerase chain reaction technique was used to detect and quantify
gene expression (i.e. mRNA) (Keller et al., 2001). Levels of IL-6 transcription and
mRNA on the nuclei and total RNA were measured from muscle biopsies obtained
pre-, during, and post non-damaging exercise. Participants performed a two-legged
dynamic knee extensor exercise (50–60% of maximal workload) for 180 min, as
well as being on a diet regimen eliciting either normal (control) or low (~60% of
normal) muscle glycogen levels (Keller et al., 2001). Muscle biopsies prior to exer-
cise observed that the nuclear transcription gene for IL-6 was nearly undetectable,
with a rapid and pronounced increase in the transcription rate of IL-6 observed post
exercise at the nuclear level by the reverse transcription-polymerase chain reaction
technique (Keller et al., 2001; Malm et al., 2000). In response to a reduction in
muscle glycogen concentration, a greater rise in the transcription for the IL-6 gene
after 90 min (~40-fold) and 180 min (~60-fold) of exercise was observed (Keller
et al., 2001). In addition, a significant increase in IL-6 mRNA (>100-fold) was
observed after 180 min of exercise vs. control (~30-fold) suggesting that besides the
type of muscular contraction (i.e. eccentric or concentric), concentration of muscle
glycogen is also critical in the release of IL-6 during habitual exercise (Keller et al.,
2001; Steensberg et al., 2001).
Muscle as an Endogenous Glucose-Producing Organ
Skeletal muscle is considered an endogenous glucose-producing organ influencing

the disposal of glucose, with a greater increase in glucose uptake occurring during
contraction compared to the maximal stimulation by insulin (Febbraio, Hiscock,
Sacchetti, Fischer, & Pedersen, 2004). Considering that IL-6 is produced in the
absence of inflammatory markers, and that expression of intramuscular IL-6 mRNA
is exacerbated in response to compromised glycogen concentration, expression of
IL-6 due to glycogen content suggests that skeletal muscle plays a metabolic role as
well (Pedersen & Febbraio, 2008). A study evaluating the metabolic effects of IL-6
for treatment of metastatic renal cell cancer observed that IL-6 produced an endo-
crine response in patient’s administered recombinant human IL-6 (Stouthard et al.,
1995), with a greater increase in hepatic glucose occurring on the recombinant
human IL-6 infusion vs. the control (Stouthard et al., 1995). In agreement with the
previous study, a study that administered IL-6 subcutaneously to healthy volunteers
noticed an increase in glucose (Tsigos et al., 1997). The release of glucagon induced
by IL-6 increased hepatic glycogenolysis contributing to elevations of blood glu-
cose (Tsigos et al., 1997). An in vitro study designed to investigate whether radioac-
tive glucose released from cultured hepatocytes consisted of radioactive pre-labelled
glycogen pools observed that IL-6 was a strong glucoregulatory cytokine stimulat-
ing the release of hepatic glucose (Ritchie, 1990). Interestingly, in the past, IL-6 was
once referred to as a hepatocyte-stimulating factor (Heinrich et al., 1998).
According to Gleeson (2000), the release of IL-6 in lieu of glucose concentration
and contracting muscle suggests that this myokine acts as a hormone mediating the
hepatic glucose output necessary for maintaining the homeostasis of glucose during
non-damaging exercise. Therefore, since skeletal muscle is considered an endocrine
organ, responsible for the production, release, and expression of several cytokines,
an important link has been established between skeletal muscle, metabolic changes,
and the modification of cytokine production in tissue and organs in response to
habitual exercise (Pedersen, 2006; Pedersen et al., 2001).
Intensity, Duration, and Mode of Exercise
The extent to which the muscle is activated depends on the intensity, duration, and
the mode of exercise (i.e. the amount of muscle mass recruited) (Pedersen &
Febbraio, 2008). A study investigating the importance of intensity and the release of
IL-6 had seven untrained healthy male participants perform a 45-min knee exten-
sion exercise with both legs kicking at a frequency of 60 kicks per minute at 25%
Wmax, on two independent parallel one-leg knee extension ergometers, simultane-
ously (Helge et al., 2003). The blood was collected from both the femoral artery and
veins at 15, 30, and 40 min. After 45 min, the exercise was stopped and the load was
adjusted. When exercise resumed for another 35 min, one leg performed a knee
extension exercise kicking at 65% Wmax (moderate intensity), with the other kicking
at 85% Wmax (high intensity). The blood was sampled at 15, 30, and 35 min, with a
muscle biopsy taken pre- and post-80 min of exercise. The main finding of the study
was that the release of IL-6 by the working muscle was related to both the intensity
of the exercise and the glucose uptake (Helge et al., 2003), confirming that the
release of IL-6 by the contracting muscle is important for maintaining glucose
homeostasis during habitual exercise (Gleeson, 2000). Interestingly, although the
release of IL-6 by the contracting muscle is prompted by the need to supply fuel,
this myokine also accommodates the need for fuel by stimulating lipolysis in adi-
pose tissue in response to diminished muscular carbohydrate (Helge et al., 2003).
Besides intensity, duration of exercise is associated with release of IL-6. A study
investigating the effect of a prolonged one-legged dynamic knee extensor exercise
with six healthy males for 5 h at a power output of 25 W, representing 40% of their
peak power output (Wmax), observed an increase in production and high turnover of
IL-6 (Steensberg et al., 2000). Participants moved the ankle over a range of ~60°
(from 90 to 30° angle), with the blood collected before and after each hour of exer-
cise from both the femoral artery and vein of the exercising leg and the femoral vein
of the resting leg. Over the last 2 h of exercise, a release of IL-6 from the muscle was
17-fold higher than the arterial concentration (Steensberg et al., 2000). The effects
of a prolonged exercise in the form of marathon running (Copenhagen Marathon)
also measured an increase in IL-6 mRNA locally in activated skeletal muscle
(Ostrowski et al., 1998). Blood samples were drawn from the antecubital vein of 16
male marathoners 1 week before, immediately after, and 2 h postrace, with similar
time periods for muscle biopsies (vastus lateralis) taken from eight of the par-
ticipants. The expression of mRNA was not observed in the circulating blood
mononuclear cells suggesting that no contribution occurred from muscle damage.
Inflammation 65
In addition, although increases in plasma IL-6 was observed postexercise, its decline
thereafter coincided with an increase in IL-1ra (anti-inflammatory cytokine) con-
centration 2 h postexercise (Ostrowski et al., 1998), suggesting that acute exercise
creates an IL-6 anti-inflammatory environment.
Another source for the release of IL-6 is the mode of exercise, with the mass of
skeletal muscle recruited during exercise influencing the expression of the cyto-
kines. A study in which ten experienced triathletes performed two cycling and run-
ning sessions spread out over a 4- to 6-week period, observed that running (eccentric
exercise), which recruited more muscle mass, had a greater release in IL-6 versus
cycling (concentric exercise) (Nieman et al., 1998). Similarly, this observation was
supported by a study that observed a pronounced increase in systemic concentration
of IL-6 occured during running vs. cycling (Febbraio & Pedersen, 2002). In conclu-
sion, circulating levels of IL-6 in response to habitual exercise occur without muscle
damage, with this rise in concentration influenced by the intensity, duration, and the
amount of muscle mass recruited (Jonsdottir et al., 2000; Pedersen & Fischer, 2007;
Steensberg et al., 2000; Ullum et al., 1994).
Damaging Exercise and the Inflammatory Response
Since the 1960s, sports scientists observed that small lesions in muscle structure
expressing a small foci of inflammation and degenerative changes, including fibre
necrosis, were characteristic of a single bout of exhaustive exercise – either short-
lasting intensive or long-lasting moderate (Vihko, Rantamaki, & Salminen, 1978).
This disruption is associated with crush and strain injuries, overloading, and eccen-
tric exercise, responsible for initiating a sequence of cellular responses, expressing
an inflammatory response (Tidball, 1995). Unlike the cytokine cascade observed for
habitual exercise, the release of IL-6 is preceded by TNF-α, with the order of release
occurring as follows: IL-1β, TNF-α, and IL-6. It should be noted that IL-6, classi-
fied as an “inflammation-responsive” cytokine, does not directly cause inflamma-
tion, even though its infusion has resulted in fever in humans (Mastorakos, Chrousos,
& Weber, 1993). In addition, unlike IL-1β and TNF-α, it is not associated with
capillary leakage or shock, the upregulation of nitric oxide or matrix metallopro-
teinase, which are all major inflammatory mediators stimulated by both IL-1 and
TNF-a (Barton, 1997; Mastorakos et al., 1993). As mentioned above, IL-6 is a pri-
mary inducer of C-reactive protein and for the synthesis of cytokine inhibitors
(IL-1ra, soluble TNF-α receptors, and IL-10).
With muscle damage, the sequence/time response of the cytokines of the local
inflammatory response, is firstly pro-inflammatory in nature, with the release of
IL-1β and TNF-α expressed in skeletal muscle (Cannon & St. Pierre, 1998), fol-
lowed by the expression of the anti-inflammatory cytokines IL-6, IL-1ra, soluble
TNF-α receptors, and IL-10 (Fig. 2.18). Although cytokines are classified as either
pro- or anti-inflammatory, this classification is quite simplistic given that some cyto-
kines (i.e. IL-6) may act as either (Cavaillon, 2001). According to Cavaillon (2001),
the nature of the activating signal, the sequence and timing of action, and the nature
Fig. 2.18 Cytokine cascade related to muscle damage. (Modified from Pedersen 2006)
Fig. 2.19 The four stages with regard to muscle-damaging exercise and inflammatory response.
(Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights
reserved)
of the target cell, greatly influences the properties of the cytokines and their
subsequent expression as either pro- or anti-inflammatory in nature.
Stages Associated with Muscle Damage Exercise and Inflammation
The series of mind maps (Figs. 2.19, 2.20, 2.21, 2.22, 2.23, 2.24, 2.25, and 2.26)
and Table 2.1 below suggest a road map concerning acute inflammation and muscle-
damaging exercise. These “stages” and Table 2.1 were created to provide a logical
sequence of events, in an attempt to simplify the complex processes associated with
muscle-damaging exercises, acute inflammation, and muscle regeneration. These
Inflammation 67
Fig. 2.20 Stage1: Muscle damage. (Published with kind permission of Copyright © Nikos
Fig. 2.21 Stage 2: Autolytic calpain. (Published with kind permission of Copyright © Nikos
Fig. 2.22 Stage 3: Adhesion and transendothelial migration of leukocytes. (Published with kind
permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)
Fig. 2.23 Stage 4: Inflammatory cells. (Published with kind permission of copyright © Nikos
Fig. 2.24 Stage 4: Inflammatory cells—neutrophils. (Published with kind permission of copyright
Fig. 2.25 Stage 4: Inflammatory cells—pro-inflammatory macrophages (phagocytic). (Published

with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)
Inflammation 69
Fig. 2.26 Stage 4: Inflammatory cells—anti-inflammatory macrophages (non-phagocytic).

(Published with kind permission of copyright © Nikos C. Apostolopoulos 2018. All rights
reserved)
Table 2.1 Time scale of inflammatory cell response in relation to the phases of muscle regeneration
post muscle damage (Published with kind permission of copyright © Nikos C. Apostolopoulos
2018. All rights reserved)
Time scale – muscle damage (inflammatory cell response)
0h 2–24 h 24 h–2 days 2–4 days 4–10 days 7–10–15 days 15–20 days
Neutrophil invasion
Degeneration/necrosis Phagocytic macrophage invasion
Phases of muscle regeneration
Neutrophil invasion
Phagocytic macrophage invasion
Inflammation Non-phagocytic macrophage invasion
Satellite cell activation
Non-phagocytic macrophages
Invasion
Regeneration Satellite cell activation
Regenerating muscle fibres
Regenerating muscle fibres
Remodelling Remodelling connective tissue
Maturation of the regenerated myofibres
Rescue of the functional
performance of injured muscle
Maturation/functional repair Innervation of regenerated
fibres
“stages” are not definitive in nature but rather suggestive (but evidence-informed),
since the complex processes affiliated with acute inflammation [pro-inflammatory
myocytes in circulation, transmigration and adhesion molecules, cytokines (pro-
and/or anti-inflammatory, etc.)] overlap and are not isolated incidents. Based on
collective research findings, four “stages” have been identified and mapped. The
first stage refers to muscle damage, the cause responsible for the loss of the organ-
isation of the sarcomere, with the second stage referring to the autolytic calpain,
referencing the disruption of cellular Ca2+ by a mechanical stimulus, expressing the
upregulation of calpain (calcium-activated proteases). This protease is involved in
the autolysis of the muscle components. The third stage, adhesion and transendo-
thelial migration (TEM) of leukocytes, refers to activation of the endothelium
responsible for attracting the leukocytes (i.e. neutrophils) to the site of inflammation,
and finally, the fourth stage, the inflammatory cells. This last stage references the
neutrophils, as well as the pro- and anti-inflammatory macrophages activated at the
site of inflammation.
Stage 1: Muscle Damage (Fig. 2.20)

The catalyst for inflammation is the loss of normal sarcomeric organisation, due to
the retraction of the myofibrils, in response to exercise-induced muscle damage
(Tidball, 1995). The first study referring to morphological disruptions of the muscle
in humans, had five healthy males rapidly run down ten flights of stairs for ten
times, with the rest period consisting of taking the elevator to the tenth floor (Friden,
Sjostrom, & Ekblom, 1981). Muscle biopsies obtained from the right and left soleus
2 weeks prior and on the second and seventh day postexercise, were divided into
two halves, with one-half prepared for electron microscopy, and the other for
enzyme histochemistry. At the cellular level, muscle fibres prior to and postexercise
appeared normal, tightly packed, in well-organised fascicles, suggesting no signs of
ischemic fibre necrosis or rupture in the sore muscles. However, at the subcellular
level, focal disturbances of the characteristic cross-striated band pattern of the mus-
cle was observed 2 days postexercise, with disturbances estimated to being three
times greater comparable to sections from both control and 7 days postexercise. The
Z-disc showed marked “broadening” and “streaming” (structural disruption of the
material of Z-disc ripped out across a large portion of the sarcomere) with the myo-
filamentous material in the adjacent sarcomeres being either supercontracted or dis-
organised. In addition, the normally regular and complex fine structure of the
Z-discs had gaps in their characteristic lattice pattern. The high myofibrillar tension
developed during activation of the contractile material (interdigitating arrays of the
thick and thin myofilaments) accounts for this mechanical disruption in the Z-discs,
suggestive of a potential weak link in the contractile chain of the myofibres (Friden
et al., 1981).
A study suggesting that muscle damage following eccentric work is mechani-
cally induced, with further disruption caused by mechanical and biochemical fac-
tors, had four healthy normal participants perform a 20-min step test (Newham,
Mcphail, Mills, & Edwards, 1983). The quadriceps group of one leg performed a
concentric (stepping up) with the other an eccentric (stepping down) contraction,
each lasting 1 s at a stepping frequency of 15 cycles/min (Newham, Mcphail, et al.,
1983). Biopsies for three participants were taken immediately prior to exercise, with
further biopsies taken immediately, and at 24 and 48 h postexercise, with the latter
times coinciding with pain and tenderness of the eccentrically contracted quadri-
ceps [delayed onset muscle soreness (DOMS)]. Biopsies prepared for electron
microscopy observed areas of myofibrillar disruption. These disruptions were
counted and classified as either “focal” (areas affecting one or two adjacent myofi-
brils and one or two adjacent sarcomeres), “extensive” (areas affecting more than
two adjacent myofibrils and sarcomeres or a fibre containing more than ten focal
areas), or “very extensive” (areas containing more than one extensive area of damage).
Inflammation 71
No abnormalities were observed in the internal architecture of the concentrically

activated quadriceps, either pre- or postexercise.
Abnormalities in the eccentric contracted quadriceps immediately postexercise
continued, with changes observed 1–2 days later. Histologically 59% appeared
“normal”, with 16% and 8% expressing “focal” and “extensive”, and “very exten-
sive” disruptions, respectively. In samples taken approximately 30 h postexercise,
45% appeared “normal”, with 6%, 23%, and 28% revealing “focal”, “extensive”,
and “very extensive” changes, respectively, disclosing that the myofilaments of the
sarcomeres in the eccentric exercise quadriceps immediately and at 30 h postexer-
cise were disorganised with the Z-disc material “streaming” across the sarcomere
(Newham, Mcphail, et al., 1983).
With damage varying based on the time period, more sarcomeres exhibited
greater and extensive damage at 30 h post-eccentric exercise (Fig. 2.27). This time
course of morphological changes, with more damage observed considerably later in
the postexercise period vs. immediately post, suggests the delayed release of cre-
atine kinase (muscle damage biomarker) into circulation, peaking 4–5 days postex-
ercise (Newham, Jones, & Edwards, 1983). Interestingly, a study investigating the
effects of passive stretching in relation to an immobilised soleus muscle of male
Wistar rats, observed that stretching was also responsible for morphological changes
to the muscle fibre (Gomes, Cornachione, Salvini, & Mattiello-Sverzut, 2007).
Ultrastructural examination observed a disruption of the sarcomere, with the myofi-
brils appearing fragmented at the Z-disc, prompting the conclusion that passive
stretching needs to be applied carefully in order to prevent muscle damage (Gomes
et al., 2007).
A rapid invasion of inflammatory cell populations lasting from days to weeks,
initiated by muscle damage, is instrumental in promoting further disruption in mus-
cle homeostasis or repair (Tidball, 2005). The dead or dying cells release inflamma-
tory mediators, cytokines, as well as chemokines, inducing chemotaxis (movement
of an organism or cell in response to a chemical stimulus) in nearby responsive cells
Fig. 2.27 Measurement of histological changes. (Published with kind permission of copyright ©
(Shek & Shephard, 1998). The injury-specific interaction between the invading
inflammatory cells and muscle, the previous history of muscle use (i.e. repetitive
injuries vs. acute, eccentric vs. concentric exercise), and the magnitude of response,
determines whether the overall inflammatory response will be beneficial or detri-
mental (Tidball, 2005). In other words, the size and nature of the disruption, and the
sequencing, timing, and concentration levels of pro- and anti-inflammatory cyto-
kines, are responsible for the continued disruption or recovery of the muscle.
Stage 2: Autolytic Calpain (Fig. 2.21)

Studies referring to humans, intact animals, isolated muscle, and single muscle
fibres, have repeatedly shown that the primary site of damage is within the muscle
fibre itself (Allen, 2001). Lesions, referred to as micro-injuries, that are usually sub-
cellular, occur in small proportions in response to relatively intense, long duration,
or eccentric exercise (Armstrong, Ogilvie, & Schwane, 1983). Regardless of the
stimulus (i.e. crush injury, muscle strain, inflammation), this damage initiates a
sequence of cellular responses caused by a mechanical disruption, preceding a bio-
chemical response (Faulkner, Brooks, & Opiteck, 1993). Although mechanical dis-
ruption can explain a reduction in force production, the increased expression of
inflammatory cells is responsible for an additional decrease in function (Faulkner
et al., 1993; Macintyre, Reid, Lyster, Szasz, & Mckenzie, 1996; Tidball, 2002). The
extensive disruption of the structural components of the muscle, expressed as a loss
of normal sarcomere organisation with a retraction of the myofibrils from the injured
site, results in a structurally intact sheath surrounding the damaged region, consist-
ing of a basement membrane and endomysium proteins (Faulkner et al., 1993;
Tidball, 1995). This disruption, a disturbance of the cross-striated band pattern
affecting the myofibrillar Z-disc (streaming and broadening), involves all or a few
of the sarcomeres, either in series or parallel (Friden, Sjostrom, & Ekblom, 1983).
Ultrastructural damage involves the crumpling of the interface between the thin and
thick filaments, since their overlap is an integral component of the myofilament
structure (Brown & Hill, 1991; Higuchi, Yoshioka, & Maruyama, 1988; Horowits,
1992). Further, the disruption at the level of the Z-disc is associated with both mito-
chondrial and sarcoplasmic reticulum vacuolisation, an adaptive physiological
response to numerous environmental changes limiting damage (Belcastro, Maclean,
& Gilchrist, 1985; Friden et al., 1983). It has been suggested that vacuolisation, a
distinct form of cell death, is a degeneration resulting in lytic responses (Henics &
Wheatley, 1999).
Catabolic events associated with muscle damage involve the autolysis of compo-
nents (Armstrong, 1990; Huijbregts, 2001), affiliated with the disruption of and the
homeostasis of cellular Ca2+ (Armstrong, 1990; Belcastro, Shewchuk, & Raj, 1998),
which is released from intercellular stores that gain entry from the extracellular
space (Spencer, Lu, & Tidball, 1996). This disruption is characterised by a derange-
ment of the sarcomere, fragmented or swollen sarcoplasmic reticulum elements and
mitochondria, and lesions in the plasma membrane (Belcastro et al., 1998). The
Ca2+ molecule is important for triggering muscle contraction, relaxation, and
Inflammation 73
controlling the energetics of the muscle, by regulating the provision of ATP
(Berchtold, Brinkmeier, & Muntener, 2000; Tate, Hyek, & Taffet, 1991). Within the
myofibril, a variety of Ca2+-binding proteins (i.e. calmodulin, calpains), not involved
directly in the process of contraction and relaxation exist, which are very important
for muscle plasticity and performance (Berchtold et al., 2000; Suzuki, Hata,
Kawabata, & Sorimachi, 2004). With the release and upregulation of Ca2+ occuring
in response to the disruption of the sarcomere, and the muscle’s inability to buffer
it, the “calcium-dependent protease” calpain is expressed and upregulated (Tidball,
1995). This Ca2+ binding protein, an inactive enzyme found within the cytosol of
skeletal muscle (Kumamoto et al., 1992), is activated in the presence and increase
in Ca2+ (Suzuki et al., 2004). When activated, selective proteolysis of various struc-
tural, metabolic, and/or contractile elements (Belcastro et al., 1998), hydrolysing
enzymes (phosphatases and kinases), and cytoskeletal and membrane pro-
teins occurs (Kunimatsu, Higashiyama, Sato, Ohkubo, & Sasaki, 1989). Although
this proteolysis can occur at both the Z- and I-disc regions of the sarcomere, a
higher propensity happens to occur at the Z-disc (Kumamoto et al., 1992),
with prominent muscle proteins, including myofibrillar and major Z-disc proteins,
and those involved with myofibril linkage to cell membranes (i.e. talin and vinculin)
being cleaved (Takahasi, 1990). This calpain at the Z-disc suggests that this area of
the muscle is the most susceptible to degradation (Dayton, Reville, Goll, & Stromer,
1976). The protease activity of calpain has been observed during myofibrillar deg-
radation, with the loss of Z-disc proteins seen in 22% of the myofibrils isolated from
the skeletal muscle of exercising rats (Belcastro, Parkhouse, Dobson, & Gilchrist,
1988; Goll, Dayton, Singh, & Robson, 1991). Calpain cleaves troponin, tropomyo-
sin, α-actinin, nebulin, titin, desmin, the sarcolemmal-associated spectrin complex
of proteins, and membrane adhesion molecules (integrin, cadherin, N-CAM)
(Belcastro et al., 1998; Goll, Thompson, Li, Wei, & Cong, 2003). Since its action is
rather disruptive, destabilising and altering the substrate proteins makes them more
susceptible to various cellular proteases (Saido, Sorimachi, & Suzuki, 1994) (Fig. 2.28).
Eccentric exercise is associated with activation of calpain, since levels of intra-
cellular Ca2+ remain slightly elevated above normal resting cytoplasmic levels for
24–48 h (Murphy, 2009). Following bouts of strenuous exercise, the proteolysis of
skeletal muscle proteins by calpain (Belcastro et al., 1998) is linked to an increase
in leukocyte populations (Camus et al., 1992; Field, Gougeon, & Marliss, 1991;
Gabriel, Schwartz, Steffens, & Kindermann, 1992), for the peptides released are
chemoattractive for neutrophils without producing any cytotoxic effects (Kunimatsu
et al., 1989, 1993, 1995).
A study designed to investigate the relationship between calpain-like protease
and neutrophil accumulation, measured accumulations of neutrophil through the
activity of myeloperoxidase (MPO) (Raj, Booker, & Belcastro, 1998), an enzyme
used to measure and determine neutrophil migration into muscles (Morozov,
Tsyplenkov, Goldberg, & Kalinski, 2006). To measure this relationship, 15 male
Wistar rats were randomly assigned to either a control (n = 5) or exercise (n = 5)
group, with another exercise group (n = 5) investigating the extent of how calpain
promotes neutrophil accumulation, with the latter group pre-injected with a cysteine
Fig. 2.28 Subsequent steps associated with the autolysis by calpain. (Published with kind permis-
sion of copyright © Nikos C. Apostolopoulos 2018. All rights reserved)
Inflammation 75
protease inhibitor (reduces activity of calpain-like protease) 1 h prior to exercise.

With speed of the motorised treadmill set at 25 m/min and at an 8% grade, the rats
ran on the treadmill for 60 min or until voluntary termination. Postexercise rats,
matched for control and exercise were euthanised with blood and muscle tissue
(plantaris and ventricles) collected. Determination of calpain-like and MPO activity
was performed on serial samples from the same muscle, with plasma creatine kinase
also being measured to determine muscle damage. A major observation was the
positive relationship between the activity of the calpain-like protease and MPO for
all the studied tissues (plantaris and ventricles). More importantly, a link between
the underlying processes with skeletal muscle calpain-like and MPO activity was
observed showing a responsiveness to an exercise stimulus (Raj et al., 1998). Rats
injected with the cysteine protease inhibitor were associated with a lower MPO,
suggesting that neutrophil accumulation is dependent upon Ca2+-stimulated cysteine
proteases (Raj et al., 1998).
Since the release of the peptides by the activity of calpain was chemoattractive
for neutrophils (Kunimatsu et al., 1989), this signifies the shift from “stage 2” to
“stage 3” (Fig. 2.19). With the aforementioned study by Raj et al. (1998), although
the underlying mechanism regarding the enhanced responsiveness to exercise stim-
ulus of the plantaris vs. cardiac muscle of calpain-like activity and MPO is unknown,
they suggest that the synthesis of calpain is possibly related to the greater force
production (intensity) generated by the plantaris muscle. Skeletal muscle contrac-
tion is associated with a greater metabolic demand dependent on the Ca2+-
homeostasis, with eccentric exercise linked to a continued degradation of myofibrillar
protein, a metabolic effect, affiliated with an extensive delay vs. immediate damage
(Evans & Cannon, 1991). Interestingly, elevation of muscle Ca2+ observed in female
Sprague-Dawley rats following stretching suggests that stretching may be a poten-
tial mechanism for disruption of the homeostasis of Ca2+ (Armstrong et al., 1993).
Stage 3: Adhesion and Transendothelial Migration (TEM) of Leukocytes

(Fig. 2.22)
Recruitment of leukocytes to an injured site is a hallmark of the inflammatory
response, for “no immune response”, either innate or adaptive, can occur unless
leukocytes cross the blood vessels. This complex multistep process, with each step
being a prerequisite for the next, consists of many adhesion molecules on both leu-
kocyte and endothelial cell surfaces consisting of overlapping functions (Schenkel,
Mamdouh, & Muller, 2004) (Fig. 2.29). When skeletal muscle is damaged, leuko-
cytes, specifically neutrophils, are attracted to the site of injury through the process
of adhesion and transendothelial migration. In vitro (cultured endothelium) and
in vivo (animal intravital microscopy) studies suggest that transendothelial migra-
tion is a critical step in the regulation of the inflammatory response, contingent upon
leukocytes crossing the endothelial lining of the blood vessels to enter the site of
inflammation (Kim & Luster, 2015; Muller, 2013). It is important to note that regu-
lation of the inflammatory response is very critical, for beneficial and collateral
damage occurs once the leukocytes leave circulation.
Fig. 2.29 Transendothelial migration. (Published with kind permission of copyright © Nikos
The largest pool of circulating leukocytes in the bloodstream, the neutrophils

(40–80%), persist for hours to days until they reach senescence and are cleared from
the body (Kim & Luster, 2015). Because of their sheer volume, they are often mar-
ginalised towards the vascular wall by collision with red blood cells, a process
responsible for their adherence to the vascular endothelium (Sundd, Pospieszalska,
& Ley, 2013). Since it is beyond the scope of this manuscript to describe the intrica-
cies of adhesion and transmigration, readers are directed to the “Further Readings”
section at the end of the chapter.
Before adhesion and transmigration can take place, leukocyte recruitment is ini-
tiated by the release of a chemoattractant agent, such as the release of the peptides
by the activity of calpain, as a chemoattractant agent for neutrophils (Kunimatsu
et al., 1989). The importance of the release of this agent was investigated by a study
observing responses of leukocytes to skeletal muscle damage (i.e. crush injury) of
the mid-region of the tibialis anterior of female inbred Swiss mice at zero (0), three
(3), and 24 h post crush injury vs. non-injured muscle (Robertson et al., 1993). A
chemotactic response occurred at both 3 and 24 h post damage, with migrating leu-
kocytes accumulating in clumps near regions where muscle fragments were pro-
nounced. Although an accumulation of neutrophils occurred at 3 h, a far stronger
chemotactic response was observed for exudate vs. resident macrophages within
24 h (Minutti, Knipper, Allen, & Zaiss, 2017; Robertson et al., 1993). No chemoat-
tractant response was present for macrophages in normal uninjured skeletal muscle,
nor muscle removed immediately post crush (0 h) (Robertson et al., 1993).
Considering that neutrophils and macrophages were expressed in injured muscle
several hours post damage (3 and 24 h), and not immediately after, suggests the
Inflammation 77
activation of resident fibroblasts and macrophages. Normally residing in a quiescent

state in the endothelium, resident macrophages and the activation of the endothe-
lium attract and activate inflammatory cells during the early stages of muscle injury
(Tidball, 1995).
During homeostatic conditions, the endothelial cell layer lining the vascular
lumen, serves as a barrier limiting the infiltration of leukocytes into tissues; how-
ever, during injury, the inflamed vessel wall selectively recruits leukocytes in
response to various stimuli (Kim & Luster, 2015). In a sequence of adhesive steps,
leukocytes attach to the endothelial wall of the blood vessel and roll along the wall
to the endothelial borders traversing the endothelium, migrating through the inter-
stitial tissue into the site of injury (Ley et al., 2007; Muller, 2013). Commencement
of this process is reliant upon the release of the pro-inflammatory cytokines respon-
sible for activating the endothelial cells. This initiates a well-orchestrated series of
adhesive interactions, with the increased expression of adhesion molecules (E- and
P-selectins) and integrin ligands, responsible for recruitment of neutrophils (Kim &
Luster, 2015; Puri et al., 2005). The enhancement of the E- and P-selectin on the
endothelial surface during inflammation act as receptors for the recruitment of leu-
kocytes into the site of damage (Mitroulis et al., 2015), with expression of E-selectin
inducing the synthesis of TNF-α (Cannon & St. Pierre, 1998; Weller, Isenmann, &
Vestweber, 1992). Activation of the endothelium results in a release of IL-1β, IL-6,
and IL-8 (Detmers et al., 1991; Liu & Spolarics, 2003), with induction of IL-8 by
IL-1β promoting endothelial adhesion and the chemotaxis for neutrophils (Colditz,
Zwahlen, Dewald, & Baggiolini, 1989; Willems, Joniau, Cinque, & Van Damme,
1989), with blocking of IL-1β activity using IL-1βra, reducing production of IL-8
by 85% (Porat, Poutsiaka, Miller, Granowitz, & Dinarello, 1992). Expression of
IL-1β and TNF-α in response to active stretching during eccentric exercise, has been
observed to augment leukocyte adhesion molecules on human endothelial cells
(Bevilacqua, Pober, Mendrick, Cotran, & Gimbrone, 1987; Malm et al., 2000).
The primary step of capture, rolling, and slow rolling, which enables the adher-
ence of leukocytes to the endothelium under conditions of blood flow, is dependent
upon the rapid reversible bonds between the selectin family of adhesion molecules
and the neutrophils (Ley et al., 2007). Interestingly, the shear stress of blood flow is
a requisite step for adhesion, strengthening the bond between the selectins and the
neutrophils, for when the flow is stopped, the cells detach. The E- and P-selectins
are vital for capture and rolling, for mice lacking their expression exhibit more than
a ten-fold elevated rolling velocity (Puri et al., 2005).
The processes of rolling and slow rolling bring the neutrophils into contact with
the endothelial cells, whereupon, they can be activated further by pro-inflammatory
(IL-1 and TNF-α) agents and chemokines on the surface of the endothelial cells
(Muller, 2013). The chemokines, contacting the chemokine receptors on the leuko-
cytes, activate the leukocyte integrins (Mitroulis et al., 2015). As mentioned above,
integrins are heterodimeric adhesion receptors (α/β) with most of them binding to
ECM proteins (β1 integrin) (Hynes, 1992). However, β2 integrin is associated with
leukocytes whose ligands are the intracellular adhesion molecules 1 and 2 (ICAM-1
and ICAM -2) (Hynes, 1992; Muller, 2013). Their activation results in a conformational
change favouring binding to their ligands expressed on the endothelial cells of the
inflamed endothelium. Once activated, the integrins of the leukocytes bind tightly to
their ligands on the endothelial cells, thereby allowing leukocytes to arrest on the
endothelial surface (Muller, 2013). Mice who are deficient in β2 show an increased
leukocyte rolling velocity suggesting that this integrin contributes significantly to
the “slowing-down” of rolling neutrophils (Dunne, Ballantyne, Beaudet, & Ley,
2002; Muller, 2013).
Binding of integrins to ligands triggers the “outside-in signalling” pathway
strengthening adhesion of leukocytes, but more importantly facilitating the forma-
tion of focal adhesion and further integrin-dependent steps (Mitroulis et al., 2015).
Prior to the final step of transendothelial migration, leukocytes are engaged in the
process of locomotion following firm arrest. This process is dependent upon the β2
integrin during which the leukocytes crawl on the surface layer in order to identify
an appropriate site on the endothelium to migrate through its monolayer. Locomotion
ensures that the leukocytes move efficiently to the interendothelial junctions for
transendothelial migration to occur (Mitroulis et al., 2015; Phillipson et al., 2006).
The processes of leukocyte rolling, activation, adhesion, and locomotion are all
reversible; however, transendothelial migration is not (Muller, 2013). Once the
leukocyte squeezes through the endothelial cell borders in an amoeboid fashion,
crossing the endothelial cells via the process of diapedesis, this is considered as the
point of no return in the inflammatory response (Muller, 2013). By committing to
diapedesis, the leukocyte cannot go back.
Stage 4: Inflammatory Cells (Fig. 2.23)

Neutrophils (Fig. 2.24)
Neutrophils and macrophages dominate the inflammatory response, with neutro-
phils being the first responders migrating into the affected tissue. Following eccen-
tric exercise, they invade the injured skeletal muscle within several hours, remaining
for several days (Karalaki, Filli, Philippou, & Koutsilieris, 2009; Tidball, 1995). By
measuring the activity of the MPO enzyme, a large increase in neutrophil concentra-
tion in male Sprague-Dawley rat muscle was reported immediately after 1 h of
treadmill running at 0% grade at 25 m/min until voluntary termination (Belcastro,
Arthur, Albisser, & Raj, 1996). Neutrophil presence was confirmed in a study with
nine healthy sedentary untrained men performing three sets of 15 min bouts of
downhill treadmill running (negative 16% incline) at 75% HRmax, separated by
5 min rest periods (Fielding et al., 1993). Percutaneous needle biopsies of the vastus
lateralis were taken pre-, 45 min, and 5 days postexercise, with blood samples
obtained pre-, immediately post, and at 3 and 6 h and 1, 2, 5, and 12 days postexer-
cise. A relationship between neutrophil and immunohistochemical staining for
IL-1β was observed, with a significant positive correlation between neutrophil infil-
tration and the ratio of ultrastructural damage to total Z-discs (r = 0.66; P < 0.05).
At 45 min, an accumulation of neutrophils occurred, remaining elevated for 5 days
post, with immunohistochemical staining of muscle cross sections revealing a 135%
increase in IL-1β immediately post and increasing to 250% 5 days postexercise.
Inflammation 79
This association between neutrophils and IL-1 has been confirmed by other studies
as well (Figarella-Branger, Civatte, Bartoli, & Pellissier, 2003; Pedersen, Ostrowski,
Rohde, & Bruunsgaard, 1998; Philippou et al., 2012; Smith et al., 2008).
Increases in the concentration of neutrophils within 1–6 h post injury suggest
that these circulating leukocytes play a prominent role in the early expression of the
inflammatory response (Kanda et al., 2013; Orimo, Hiyamuta, Arahata, & Sugita,
1991; Papadimitriou, Robertson, Mitchell, & Grounds, 1990; Summers et al., 2010).
They are important for removal of necrotic tissue by the process of phagocytosis,
continuing the inflammatory response with the release of the pro-inflammatory
cytokines (IL-1β and TNF-α) (Cannon & St. Pierre, 1998; Smith et al., 2008).
Elevation in neutrophils has been observed with passive stretching in 4-month old
adult mice as well (Pizza, Koh, Mcgregor, & Brooks, 2002).
Neutrophils are a source for the synthesis of IL-1β, TNF-α (Dubravec, Spriggs,
Mannick, & Rodrick, 1990; Tiku, Tiku, & Skosey, 1986), and reactive oxygen spe-
cies (i.e. superoxide or hydrogen peroxide), cytotoxic molecules which can lyse cell
membranes leading to further muscle damage (Canon et al., 1991; Mittal, Siddiqui,
Tran, Reddy, & Malik, 2014; Philippou et al., 2012). Reactive oxygen species are a
double-edged sword, for in conjunction with other chemokines and growth factors,
they also participate in muscle repair (Barbieri & Sestili, 2012). According to inves-
tigators, expression of these cytotoxins is dependent on the intensity and type of
exercise responsible for muscle damage (Nieman, 1997; Tidball, 2005).
Pro- and Anti-inflammatory Macrophages (Figs. 2.25 and 2.26)

After the initial invasion by neutrophils, macrophages appear. These active inflam-
matory cells are a source for pro-inflammatory cytokines promoting the removal of
cellular debris and muscle tissue remodelling (Kanda et al., 2013). They are com-
prised of several subtypes with distinct functions, with their phenotype character-
ised by the molecular environment of the damage (McLennan, 1996; Philippou
et al., 2012). In other words, dependent on the environment, macrophages can adopt
either a pro- or anti-inflammatory phenotype (Bystrom et al., 2008; Novak & Koh,
2013). For instance, the continued presence and accumulation of neutrophils at the
inflammatory site are responsible for the presence of pro- rather than anti-
inflammatory macrophages (Pizza, Petersen, Baas, & Koh, 2005) with the former
producing reactive oxygen species, IL-1β, and TNF-α (Bencze et al., 2012) and the
latter, when activated, producing IL-10, downregulating the production of IL-12,
characteristic of the inhibition of inflammation (Bencze et al., 2012).
With the use of a specific panel of antibodies, based on the antigenicity of the
various macrophage subtypes (ED1+ monocytes, ED1+ macrophages, ED2+Ox6−
and ED2+Ox6+, and Ox6+), McLennan investigated the arrival and departure time,
as well as the specific location within the lesion of damaged muscle for the various
macrophage subtypes (McLennan, 1996). The tibialis anterior muscle of adult male
Wistar rats (n = 55) was exposed to a freeze injury using the blunt end of a stainless
steel surgical probe (3 × 4 mm) cooled in liquid nitrogen. The rats were sacrificed at
various hours (0.5, 1, 1.5, 2, 3, 5, 7, 9, 11, and 18) and days (1, 2, 3, 3.5, 4, 4.5, 5, 6,
7, 8, 11, 14, 19, and 21) post lesion of the muscle. Sections of the muscle were
prepared for analysis using antihaemopoietic cell antibodies. Within 1 h of freezing,
damage/dead fibres had swollen, differentiating them from healthy fibres. This ini-
tial event occurred uniformly throughout the lesion, with subsequent cellular events
occurring where the lesion bordered the undamaged fibres, progressively moving
from the periphery to the centre, creating a gradient of maturity. Events within the
central core were usually delayed by 2–3 days. Cell infiltration on the periphery
began by 3 h with some fibres being completely phagocytosed by 1 day. Myotube
formation was observed to occur by 2 days, with the boundary between the dam-
aged and undamaged portion becoming undiscernible by 3 weeks.
Circulating Macrophages (ED1+ Monocytes and ED1+ Pro-inflammatory)

Within an hour of damage and in response to inflammatory signals (cytokines and
chemokines), the ED1+ monocyte infiltrated the epimysium penetrating the injury
within 3 h. The primary role of this monocyte is to replenish the population of
tissue-resident macrophages during homeostasis and inflammation (Murray &
Wynn, 2011). Upon infiltration, the ED1+ monocyte is activated into ED1+ macro-
phages, preceding ED2+ macrophages and Ox6+ cells. According to Butterfield
et al., ED1+ monocytes are the predominant macrophage phenotype in circulation
transforming into the ED1+ macrophage upon activation (Butterfield, Best, &
Merrick, 2006). The presence of ED1+ cells, associated with mature lesions, clearly
indicates where damage has occured, for near the end of the degenerative phase,
ED1+ macrophages are less apparent.
Resident Macrophages (Ox6+, ED2+Ox6−, ED2+Ox6+, and ED2+)

Resident macrophages, representing up to 10–15% of the total cell number during
quiescent states in adult mammals, are versatile cells found in essentially all adult
mammal tissues, with this number increasing in response to inflammation (Italiani
& Boraschi, 2014). Concentrations of OX6+, a major subtype of resident macro-
phages, are completely absent early in the lesion, possibly in response to the death
of the resident macrophages, as the damaged area contained some OX6+ cellular
debris, with their numerical density behind the lesion being normal. Their appear-
ance in the core of the lesion always occurred after the presence of ED1+ mono-
cytes/macrophages with their numbers exceeding ED1+ cells. Increased accumulation
of Ox6+ within the necrotic fibres, indicates early stages of degeneration, being less
abundant in extensively phagocytosed fibres. As the lesion regenerated, the Ox6+
cells become more localised to the endomysium and perimysium, being initially
more abundant than ED2+ macrophages in the endomysium, with the converse for
the perimysium. They are normally located in both the endo- and perimysium, with
their concentrations being greater during regeneration than in undamaged muscles.
Three weeks post damage, their numerical density declined, making it difficult to
delineate between damaged and undamaged tissue.
Inflammation 81
According to Honda et al. (Honda, Kimura, & Rostami, 1990) and McLennan
(McLennan, 1993), ED2+Ox6− is a major type of resident macrophage in skeletal
muscles. Within lesioned areas, they appeared to die and rapidly regenerate. After
1 h post damage, the ED2+ macrophages were no longer detectable; however, they
were still abundant in undamaged portions of the muscle. At 3 h a progressive
increase in their numbers occur on the fringe, with a heightened accumulation
formed on the fringes of the lesions and the perimysia behind them by 1–2 days
(Honda et al., 1990; McLennan, 1993). It should be noted that the majority of ED2+
in the connective tissue, both behind and overlaying the lesions, were of the
ED2+Ox6− phenotype. ED2+ macrophages are not associated with necrotic fibres
during early stages of phagocytosis (Honda et al., 1990).
Spatial Sequencing of Inflammatory Cells

Evidence exists suggesting that overload, overuse, and compression induce struc-
tural damage and inflammation to both muscle and tendon tissue (Kuipers, 1994;
Messner, Wei, Andersson, Gillquist, & Rasanen, 1999; Sharma & Maffulli, 2006;
Sorichter, Puschendorf, & Mair, 1999; Soslowsky et al., 2000). A study specifically
referring to the time course of inflammatory cell accumulation in two animal mod-
els, with regard to structural damage and inflammation following a tendon injury
(acute tendinopathy), provides a summary of the spatial sequencing of the various
inflammatory cells, specifically the accumulation and decrease in the concentration
of the neutrophils, ED1+ (phagocytic) and ED2+ (non-phagocytic) macrophages
(Marsolais, Cote, & Frenette, 2001). Although this study referred to a tendon injury,
regardless of the inciting factors, the events associated with inflammation and tissue
damage are considered to be the same, with the kinetics and the magnitude of the
response dependent on the extent of the damage as well as the muscle damaged
(Butterfield et al., 2006; Carlson & Faulkner, 1983; Charge & Rudnicki, 2004;
Lefaucheur & Sebille, 1995; Tidball, 1995).
This study by Marsolais et al. (2001) consisted of female Wistar rats, and com-
prised of several models. With the first model, a blunt dissection of the right isolated
exposed Achilles tendon was performed, and injected with 30μl of crude collage-
nase dissolved in sterile phosphate buffered solution (PBS) near the osteo-tendinous
junction of 24 rats, inducing Achilles tendinitis (Davidson et al., 1997). The proce-
dure was repeated with sham-operated animals using the same volume of PBS with-
out collagenase. Ambulatory controls not injected with either collagenase or PBS
were allowed to roam freely in their cage. Since the sham group in the first model
expressed a high number of inflammatory cells (neutrophils and ED1+ macro-
phages), following the exposed Achilles tendon procedure, a second model was
setup to determine whether the surgical intervention and not the percutaneously
injected collagenase and PBS was responsible for inflammation. Rats in this model
were injected with the collagenase and PBS percutaneously without any surgical
intervention, with the same volume of PBS without collagenase being injected in
the sham animals, and with the ambulatory control rats not being injected with
either collagenase or PBS. Rats in the first model (experimental, sham, and ambulatory
control) were sacrificed at 1, 3, 7, 14 or 28 days post collagenase and PBS injection,

with the rats in the second model sacrificed after 1 and 3 days. For both models, the
sham animals were sacrificed at 1 and 3 days. A comparison of neutrophils and
ED1+ and ED2+ macrophages between the two groups (experimental and sham) for
both models was performed at 1 or 3 days post collagenase or PBS injections, with
this time period being reflective of the time encompassing an extensive cell accumu-
lation of the inflammatory cells.
The injured tendon expressed an accumulation of leukocytes with a rapid infiltra-
tion of neutrophils followed by ED1+ macrophages but to a lesser degree, with
regard to post collagenase injection. There was a 46-fold increase in neutrophil
concentration at 1 day post injury in the exposed Achilles tendon animals, decreas-
ing more than 70% after 3 days, with a return to control values at 7, 14, and 28 days
post injury. The time points used in this study (1, 3, 7, 14, and 28 days) are sugges-
tive of the phases of inflammation and its resolution (Frenette, St-Pierre, Cote,
Mylona, & Pizza, 2002) (Table 2.1). A comparison of immunohistochemistry results
revealed a high concentration of neutrophils after 1 day of the injured tendon vs.
being completely absent in the tendon from both the ambulatory controls and those
that had recovered at 28 days. Concerning ED1+, an 18-fold increase occurred at
1 day, decreasing slightly at 3 days post injury, with the most significant decrease
occurring by day 7, matching ambulatory control concentrations after 14 days.
Comparing ED1+ to neutrophils, the latter had a twofold increase in numbers after
1 day, with ED2+ values augmented after 28 days (Table 2.2). Comparing the sham
to the ambulatory control rats in the first model, a significant increase in both neu-
trophils and ED1+ concentrations was observed in the sham animals, which were
exposed to the surgical procedure minus the collagenase and PBS. This result
Table 2.2 Sequence of inflammatory cells

Time scale: Achilles tendon damage
Inflammatory cells
0–24 h 3 days 7 days 14 days 28 days
Neutrophils ↑46- fold post trauma. Highly Significant ↓ ( 70%) Return to control values post trauma. Absent
concentrated after 1 day (note: in ambulatory and animals recovered for 28
magnitude of ↑in cell number two days
Model One a
fold greater than ED1+)

ED1+ ↑ 18-fold increase ↓ Slightly ↓Significantly Similar to control
ED2+
Note: Experimental (exposed Achilles tendon and collagenase + PBS injection) > sham (exposed Achilles tendon and PBS) > ambulatory control
(no collagenase and no PBS)
Neutrophils ≈ 35% less than same time point
for neutrophils in model one
ED1+ ≈ 39% less than same time point
Model Two b
for macrophages in model one

ED2+ Significant ↑ 3 days
following injection of
collagenase
Note: no significant difference in the number of inflammatory cells between sham and ambulatory controls
a
Model one (exposed Achilles tendon) = experimental group (surgery + collagenase and PBS);
sham group (surgery + PBS), ambulatory control (no collagenase and no PBS)
b
Model two (nonexposed Achilles tendon) = experimental group (collagenase and PBS); sham
group (PBS), ambulatory control (no collagenase and PBS)
Inflammation 83
suggests that a mechanical stimulus is a major contributing factor for inducing

muscle damage and inflammation (Frenette et al., 2002; Kuipers, 1994; MacIntyre
et al., 1995).
Interestingly, the appearance of leukocytes was similar in both the nonexposed
and exposed Achilles tendon groups at 1 and 3 days, confirming that regardless of
the insult causing the damage, the sequencing of the cells of the inflammatory
response is the same (Butterfield et al., 2006; Tidball, 1995). The nonexposed
Achilles tendon procedure was associated with a decrease in the magnitude of the
leukocyte cell accumulation, ~35% for neutrophils, and ~39% for ED1+ macro-
phages. Following collagenase injection, ED2+ macrophages increased in the non-
exposed Achilles tendon group after 3 days. With both the ambulatory controls and
sham animals, which underwent the nonexposed Achilles tendon procedure, no sig-
nificant difference in the number of inflammatory cells was expressed. Based on the
results of this study (Marsolais et al., 2001), as well as several other studies, neutro-
phils and ED1+ macrophages express a phagocytic phenotype responsible for the
removal of cellular debris, with ED2+ macrophages associated mainly with the
regeneration of damaged muscle (Al-Mokdad, Shibata, & Nakagawa, 1997;
Massimino et al., 1997; McLennan, 1993, 1996; St. Pierre & Tidball, 1994).
By referring to Table 2.2, based on the Marsolais et al. (2001) study, we notice
that in model one [mechanical insult (blunt dissection with injection of collagenase
and PBS) to Achilles tendon] vs. model two [no exposure to mechanical stimulus
(blunt dissection)], the ED2+ (non-phagocytic) macrophages associated with recov-
ery accumulate earlier post collagenase injection (Table 2.2; yellow box). This
observation suggests that the degree and magnitude of muscle damage are catalysts
for the accumulation and phenotype of inflammatory proteins. As observed in stud-
ies referencing neutrophils, and pro- and anti-inflammatory macrophages, the neu-
trophils and the macrophages dominate the inflammatory response to muscle
damage, with the ED1+ and ED2+ macrophages possessing diverse roles with
regard to the inflammatory response and repair (Karalaki et al., 2009; Philippou
et al., 2012; Tidball, 1995). The ED1+ macrophages feature prominently in the
removal of necrotic tissue, with the ED2+ macrophages associated with muscle tis-
sue repair typically observed at later stages of inflammation. The early appearance
of the ED2+ macrophage suggests that the regeneration process occurs earlier with
a less traumatic insult on the tendon tissue (model 2 nonmechanical stimulus)
(Marsolais et al., 2001). Therefore, the degree of muscle damage, and the interac-
tion and coordination of the various infiltrating inflammatory cells, are important
for the outcome of the repair process of the muscle (Marsolais et al., 2001). This
observation is significant when considering that the magnitude of stretching inten-
sity may be responsible for causing inflammation as well as the recovery of the
muscle from injury.
Neutrophils and macrophages play an important role in both the initial response
to tissue damage and the subsequent resolution and regeneration of muscle fibre. At
the site of tissue damage, these inflammatory cells coexist, with their existence
dependent on their ability to perform distinct functions (Marsolais et al., 2001;
McLennan, 1996). According to Tidball et.al (1999) macrophages do not contribute
to membrane disruption during inflammation even though concentration of ED1+

(phagocytic) macrophages is observed to peak with muscle damage (Tidball et al.,
1999). However, the ability of the macrophages, specifically ED2+, to repair the
damage is quite limited in the absence of neutrophils, since the primary role of neu-
trophils is the removal of cellular debris through phagocytosis, prompting the mac-
rophages for cellular regeneration and repair (Grounds, 1987; Wynn & Vannella,
2016). On the other hand, the continued presence and accumulation of neutrophils
can impair tissue regeneration, since they can induce a secondary muscle injury
by modifying skeletal muscle proteins oxidatively (Pizza et al., 2005). Elevated lev-
els of neutrophils have also been associated with passive stretches, without any
signs of injury (Pizza et al., 2002).
With regard to the macrophages, the microenvironment in which they find itself
is essential for their expressed phenotype (ED1+, ED2+) (McLennan, 1996), with the
polarisation signals being apoptotic cells (i.e. dead neutrophils), as well as cyto-
kines released by other inflammatory cells (Butterfield et al., 2006; Duque &
Descoteaux, 2014). For instance, a microenvironment populated predominately by
necrotic tissue expresses ED1+ (Marsolais et al., 2001; McLennan, 1996). Similar to
the neutrophils, ED1+ are activated by pro-inflammatory cytokines TNF-α and
IL-1β (Hirani, Antonicelli, Strieter, Wiesener, & Ratcliffe, 2001). Once ED1+ mono-
cytes are activated to the ED1+ macrophage phenotype, they contribute to the
inflammatory response by releasing pro-inflammatory cytokines, such as prosta-
glandin-E2 and IL-1β, thereby recruiting more neutrophils, and thus heightening the
inflammatory response (Scott, Khan, Cook, & Duronio, 2004). The activated ED1+
macrophages produce nitrogen and oxygen intermediaries [nitric oxide (NO) and
superoxide] which are highly toxic and have the capacity to cause damage to neigh-
bouring tissues (Nathan & Ding, 2010). They are believed to be involved in various
chronic inflammatory diseases (Sindrilaru et al., 2011) making it very important to
control their responses in order to prevent further collateral tissue damage (Murray
& Wynn, 2011).
As observed by the studies above, the ED2+ macrophages appear during the latter
stages of inflammation (Marsolais et al., 2001; McLennan, 1996). Similar to ED1+,
ED2+ macrophages originate in the bone marrow from hematopoietic stem cells and
are expressed in circulation as anti-inflammatory monocytes, becoming resident tis-
sue macrophages (Bosurgi, Manfredi, & Rovere-Querini, 2011; Brigitte et al., 2010;
Yang, Zhang, Yu, Yang, & Wang, 2014). These represent 10–15% of the total adult
mammal cell population (Italiani & Boraschi, 2014). Unlike the preceding ED1+
macrophage, which is drawn to the necrotic tissue to phagocytose the cellular debris
and apoptotic neutrophils, the primary role for ED2+ is that of tissue repair through
cell signalling and cytokine production (Fernando, Reyes, Iannuzzi, Leung, &
Mckay, 2014; Philippou et al., 2012). Interestingly, unlike the ED1+ macrophages,
derived from the circulating monocytes and involved in severe inflammatory inju-
ries, resident macrophages are involved in their repopulation after a mild injury
(Italiani & Boraschi, 2014). These resident macrophages mediate tissue repair by
releasing a number of growth-promoting factors and cytokines, such as fibroblast
Inflammation 85
growth factor, insulin-like growth factor, and transforming growth factor (TGF)-β1
(Philippou et al., 2012; Robertson et al., 1993; Wahl et al., 1987), as well as IL-10,
an anti-inflammatory cytokine attenuating the ED1+ macrophage (Tidball & Villalta,
2010). In addition, ED2+ macrophages produce matrix metalloproteinases and
tissue inhibitors of metalloproteinases controlling the turnover of ECM (Wynn,
2008), as well as removing and digesting dead cells and debris, that would other-
wise promote the responses of the ED1+ macrophage (Atabai et al., 2009; Baron &
Wynn, 2011).
Interestingly, secretion of TGF-β1 by ED2+ signifies a shift from the ED1+ phe-
notype (inflammatory macrophage) towards the ED2+ (anti-inflammatory macro-
phage) in response to the phagocytosis of apoptotic neutrophils and necrotic muscle
cells (Arnold et al., 2007; Ashcroft, 1999; Fadok et al., 1989; Tidball & Villalta,
2010). This shift serves to resolve muscle inflammation. Apoptosis (programmed
cell death) is an energy-efficient means of removing the cellular debris, since the
cells that phagocytose the debris (i.e. neutrophils and ED1+ macrophages) do not
have to migrate and do not cause further inflammation (Brown, Kao, & Greenhalgh,
1992). More importantly, it is a necessary step in replacing cell populations in order
to begin the next phase of healing (Greenhalgh, 1998). However, if the natural apop-
totic events are tampered with, an exacerbation rather than a resolution of inflamma-
tion occurs, since the balance between the cellular numbers is lost leading to
non-normal tissue repair (Greenhalgh, 1998). Further, the cytokines, fibroblast
growth factor, insulin growth factor-1, and TGF-β1, are important for recruiting and
activating fibroblasts which begin the repair process by secreting matrix molecules
such as collagen (Butterfield et al., 2006). In both animals and humans, acute lim-
ited injury is accompanied by only a transient increase in TGF-β1, without fibrosis
occurring (Border & Noble, 1994). However, the stimulus of repeated injuries
increases the production of TGF-β1, sustaining its presence and levels, leading to
the further deposition of ECM and the increased expression of tissue fibrosis
(Chikenji et al., 2014; Zimkowska et al., 2009).
Therefore, infiltration of the leukocytes to the site of injury and inflammation,
sequencing of the neutrophils and the macrophages (pro-inflammatory preceding
anti-inflammatory), their coexistence and communication in response to the early
removal of cellular debris, the apoptosis of the inflammatory cells, and the release
of growth factors and cytokines, are essential for the resolution of tissue damage.
Any delay in the sequencing and proper function of these processes slows down the
recovery from tissue damage and is responsible for the disproportionate amount of
tissue destruction and increased fibrotic tissue (i.e. scar tissue), affecting the func-
tion of the muscle. In addition, if the clearance of apoptotic cells is defective and
there is a continued accumulation and persistence of leukocytes (i.e. neutrophils),
this is responsible for the progression from acute to chronic inflammation
(Lawrence et al., 2002). Since we are more concerned with the acute inflammatory
response with regard to stretching intensity, it is beyond the scope of this manu-
script to refer to chronic inflammation and the subsequent mechanisms related to
its expression.
Concluding Remarks for Stage Four

Neutrophils and macrophages are responsible for the release of the pro-inflammatory
cytokines IL-1β and TNF-α, with their production being responsible for the expres-
sion of IL-6 (Frost & Lang, 2005). These three cytokines form a complex network,
with the expression of each influencing the other, in an attempt to regulate the
inflammatory response (Akira et al., 1990). Interleukin-6, the mediator of the acute
inflammatory response, is responsible for the release of C-reactive protein, which
plays a prominent role as an anti-inflammatory mediator inducing the expression of
IL-1ra, as well as sTNF-receptors (anti-inflammatory cytokines) (Fig. 2.30) (Tilg
et al., 1994; Tilg, Dinaello, & Meir, 1997).
As emphasised, the microenvironment of a given tissue is responsible for trans-
formation of the macrophage monocyte into the macrophage phagocytic phenotype
(Duque & Descoteaux, 2014). Many of the cytokines capable of biasing the pheno-
type of the circulating monocyte are provided by the endothelium as well as the
surrounding lymphocytes, responsible for activating the ED1+ or ED2+ macrophages
(Gordon & Taylor, 2005; Martinez, Helming, & Gordon, 2009; Martinez, Sica,
Mantovani, & Locati, 2008). The ED1+ macrophages produce pro-inflammatory
cytokines (IL-1β, TNF-α, IL-6, and IL-12) mediating destruction of pathogens and
the removal of cellular debris, with ED2+-activated macrophages producing anti-
inflammatory cytokines (IL-10, TGF-β1, and IL-6), associated with repair (Fernando
et al., 2014; Flynn, Chan, & Lin, 2011). IL-6 possesses a Janus-like nature, for
based on the surrounding microenvironment, it determines the macrophage pheno-
type (ED1+, ED2+), with its absence preventing recovery (Fernando et al., 2014;
Kopf et al., 1994; McLennan, 1996). It is necessary for the resolution of inflammation
Fig. 2.30 Expression of anti-inflammatory cytokines. (Published with kind permission of copy-
right © Nikos C. Apostolopoulos 2018. All rights reserved)
Mechanotransduction 87
as observed in mice deficient in IL-6 (IL-6−/−) (Kopf et al., 1994). Although these
mice (IL-6−/−) did not express any developmental abnormalities, they were unable
to generate an acute phase response (Kopf et al., 1994), suggesting that its induction
is an important in vivo distress signal coordinating the activities of the hepatocytes,
macrophages, and lymphocytes (Kopf et al., 1994). The relationship between IL-6
and C-reactive protein represents the body’s mechanism for resolving the acute
inflammatory response.
Mechanotransduction
Conditions under which the muscle is loaded such as during eccentric and concen-
tric exercise, or passive stretching, have been associated with the expression of cyto-
kines (Helge et al., 2003; Ostrowski et al., 1998; Pedersen et al., 2001; Pedersen &
Fischer, 2007; Steensberg et al., 2000, 2002; Ullum et al., 1994) and the elevation
of neutrophils (Frenette et al., 2002; Koh, Petersen, Pizza, & Brooks, 2003a;
Mcloughlin, Mylona, Hornberger, Esser, & Pizza, 2003; Pizza et al., 2002). The
muscle’s response to load is influenced by the parameters of intensity, duration, and
frequency (Marschall, 1999; Mujika et al., 1995), with a literature review suggest-
ing that intensity may be of greater significance (Apostolopoulos et al., 2015).
According to this review, intensity, in particular stretching intensity, is qualitative in
nature compared to the quantifiable parameters of duration and frequency, account-
ing for why stretching intensity continues to remain relatively under researched
(Apostolopoulos et al., 2015). It proposes that stretching intensity be considered as
a force or load (Apostolopoulos et al., 2015), for too little force is associated with
an elastic response with little or no gain in ROM, and too much force injuries tissue
prompting an inflammatory response (Brand, 1984; Jacobs & Sciacia, 2011;
McClure et al., 1994). The review recommends that stretching intensity, and body
position during stretching, should be considered when investigating perceived mus-
cle soreness, inflammation, and their influence on muscle health and performance
(Apostolopoulos et al., 2015, p. 275).
Exercise intensity influences the synthesis of cytokines with increases in IL-6
being observed (Ostrowski, Schjerling, & Pedersen, 2000; Peake, Suzuki, et al.,
2005). A study investigating the effect of intense running observed significant
increases in concentrations of leukocytes (neutrophils, monocytes) (Ostapiuk-
karolczuk et al., 2012). With regard to stretching intensity and performance, a study
suggests that to achieve maximum jump height athletes should perform a static
stretching intensity <50% of the point of discomfort prior to performance (Behm &
Kibele, 2007).
Many physiological functions depend on the ability of cells and tissues to sense
and react to mechanical force or load (external and internal) with their response
being essential for proper development and function (Hamill & Martinac, 2001;
Kung, 2005). For instance, cells associated with baroreceptors, proprioceptors,
spindle receptors, and Golgi tendon organs sense blood pressure, positions of limbs,
as well as muscle stretch and tension, respectively (Kung, 2005). According to
Goldspink, skeletal muscle possesses the ability to adapt its structure, function, and
metabolism in response to a mechanical stimulus (i.e. stretching and overload) and
that to a large extent this stimulus regulates the expression of individual myosin
genes (biochemical response) (Goldspink, 1999). Goldspink is alluding to the con-
cept of mechanotransduction, a process defining the response and relationship of
the cells and tissues to their environment, translating any physical input or mechani-
cal perturbation into a biochemical or biological signal (Goldspink, 1999; Hamill &
Martinac, 2001; Kung, 2005; Previtera, 2004).
Mechanotransduction defines the conversion of mechanical energy (tension,
compression, shear, etc.) into biochemical signals (Yavropoulou & Yovos, 2016). It
is considered a key regulator of many physiological processes such as, the regula-
tion of stem-cell differentiation (Engler et al., 2006), fibroblast migration (Lo,
Wang, Dembo, & Wang, 2000), the initiation of inflammation, and the triggering
of cytokine production (Makino et al., 2007; Previtera, 2004). With stretching
defined as an external and/or internal force (Weerapong et al., 2004), and the
magnitude of the force applied as the intensity of the stretch (Jacobs & Sciacia,
2011), stretching intensity may be considered as a mechanotransduction mecha-
nism, thereby influencing the inflammatory response, aiding in the recovery or fur-
ther damage of the muscle.
Mechanotransduction references the behaviour of collective interactions within
complex networks adopting a “top-down” rather than a “bottom-up” approach
(Ingber, 2008a). Rather than reverse engineer from the cellular level, it tries to
understand mechanical behaviour by implying a higher-order architecture, and how
this is influenced by physical forces (Huang, Sultan, & Ingber, 2006; Ingber, 2008a).
The main tenet of mechanotransduction is the concept of tensegrity (Ingber, 2008a).
Tensegrity is concerned with the essential maintenance of mechanical stability cre-
ated when the compression-bearing rigid structures stretch or tense the flexible
tension-bearing members. This imparts a compression on the rigid structures, which
in turn creates a counteracting force, that is equilibrated throughout the structure,
which itself is in a prestress state (Ingber, 1998, 2008a). In short, this neologism
defines the structural shape of a tensegret, as a balance attained by the interaction
between a set of members in tension and compression (Anastasi et al., 2006;
Connelly & Back, 1998). With organisms constructed as a hierarchy of systems,
tensegrity is observed at different levels from the macroscopic (bones, muscles,
tendons, etc.) to the atomic scale (Anastasi et al., 2006). In other words, each system
is comprised of its own tensional integrity, with the tensegrity at one level or size
scale composed of smaller tension/compression elements at another level, with all
levels being prestressed (Anastasi et al., 2006; Ingber, 2008a) (Fig. 2.31).
An increase in stiffness is a hallmark of prestressed structures and hierarchies,
which is essential for the mechanical responsiveness to mechanical stress or load,
itself being influenced by the intensity of the stress (McMahon, 1984). An example
of a hierarchy of tensegrity levels is the relationship between the different layers
of the musculoskeletal system, from the macroscopic to the cellular. At the
Fig. 2.31 Hierarchy of systems (macro to micro). (Published with kind permission of copyright ©
macroscopic, the stable vertical form of the body relative to the force of gravity
is maintained between the compression-bearing bones of the skeleton and the ten-
sile pull of the muscles, tendons, and ligaments (Ingber, 1998). The prestress arises
from the balance between the contractile forces generated by the muscle cells, coun-
tered by the bone matrix’s ability to resist (Ingber, 2006). Below this level, mechani-
cal force is distributed through the myofascial and ECM connections between
adjacent muscle bundles, blood vessels, and nerve tracts (Ingber, 2006). At the cel-
lular level, balance between compression and tension of the individual muscle
bundles and blood vessels is achieved between tractional forces at the parenchymal
cells, resisting the forces exerted by the stiffened ECMs surrounding the connective
tissue cells, ensuring stability (Ingber, 2006). At the Z-disc, a certain form and spac-
ing is maintained regardless of an increase in load, suggesting its ability to resist
deformation with a passive stretch (Goldstein et al., 1991). This structural criterion
allows for the ease of transmission of tension generated within the sarcomere to
bones via the tendons, for the Z-disc functions as an anchor, adjoining sets of thin
filaments end-on-end in myofibrils (Goldstein et al., 1991). This rearranging at
many size scales of the musculoskeletal system protects it against injury, since the
molecular components comprising the tensed ECMs and interconnected cytoskele-
tal elements within adherent cells, adjust accordingly to the mechanical load
imparted on the system as a whole (Ingber, 2006; Komulainen, Takala, Kuipers, &
Hesselink, 1998; Ralphs, Waggett, & Benjamin, 2002). The importance of this
architectural hierarchy is in its ability to provide a biochemical response to a
mechanical perturbation, expressed in terms of the importance of the muscle and its
ability to adapt to its immediate environment (Gans, 1991). In particular, muscle’s
effectiveness (efficiency) in terms of energy consumption and the generation of
force are facilitated by the placement of sarcomeres in fibres, and fibres into muscle,
and the role muscle plays in relation of the organism to its environment (Gans &
Abbot, 1991). This relationship of the fibres to one another, of the tendons to the
aponeuroses, and skeletal elements (bones, joints, etc.) may influence the displace-
ment of force in terms of tissue damage and response to stretching. In turn, feedback
to the magnitude and rate of force by stretching intensity on tissue may influence its
response to load and to the acute inflammatory response. In other words, since the
process of mechanotransduction is concerned with the conversion of a mechanical
perturbation into a biological signal, and stretching is a physical activity related to
force development (Weerapong et al., 2004), the intensity of the stretch (i.e. magni-
tude of force) may be linked to either the muscle’s recovery from damage or not
(Jacobs & Sciacia, 2011). Research needs to be conducted in order to investigate
this relationship.
In skeletal muscle, the contractile filaments are maintained in a highly ordered
structure by specialised proteins (i.e. actin, titin, desmin, etc.), with unaccustomed
eccentric exercise or a mechanical stimulus inducing an adverse reaction causing a
leakage of protein (creatine kinase) from skeletal muscle (Feasson et al., 2002). The
most striking morphological feature of this reaction is the extensive sarcomeric dis-
ruption and Z-disc streaming (Friden et al., 1981, 1983; Friden, Kjorell, & Thornell,
1984). A loss of this sarcomeric order of the myofibrils initiates the autolysis of the
damaged components in response to the loss of and upregulation of Ca2 (Tidball,
1995). The severity of stretching, defined as muscle fibres stretched by 50% or
greater of the optimum force generating length, is observed with a reduction in the
maximum Ca2+-activated force (contraction), with 25% of the optimum force gener-
ating length not associated with a force deficit in skeletal muscle (Balnave, Davey,
& Allen, 1997). In other words, sarcomere inhomogeneity was observed with severe
stretching (Balnave et al., 1997), with an elevation of muscle Ca2+ via an influx of
Ca2+ from the extracellular space observed during static stretching of rat soleus
muscle, suggesting a disruption of the homeostasis of Ca2+ (Armstrong et al., 1993).

This loss of and upregulation of Ca2+ is responsible for the activation of calpain
(Armstrong, 1990; Belcastro et al., 1998). This calcium-activated protease cleaves a
wide variety of myofibrillar and cytoskeletal proteins found in the muscle [α-actinin
(Takahasi, 1990), vinculin (Evans, Robson, & Stromer, 1984), including talin (Fox,
Goll, Reynolds, & Phillips, 1985), desmin and titin (Huang & Forsberg, 1998)],
with these cleaved proteins acting as chemoattractants for neutrophils (Kunimatsu
et al., 1989). A study investigating passive stretching in adult male mice observed
elevated levels of neutrophils without overt signs of injury (Pizza et al., 2002).
Therefore, since the muscle is a highly plastic tissue adaptable to various situa-
tions (i.e. physical activity, injury, stretching) (Salvini, Durigan, Peviani, & Russo,
2012), mechanotransduction may be the mechanism by which stretching, in particu-
lar stretching intensity, influences adaptation. Considering that stretching intensity,
defined as the magnitude of force or torque applied to the joint during a stretching
exercise (Jacobs & Sciacia, 2011), is responsible for stressing connective and mus-
cle tissue mechanically (Martins et al., 2013), we present the view that this is associ-
ated with a biochemical response. Muscle stretching expresses specific genes
promoting sarcomeregenesis and remodelling of the ECM in shortened and
atrophied muscles (Martins et al., 2013); it also influences the elastic and plastic
responses of the MTU, affecting its flexibility and the generation of muscle strength
(Gajdosik, 2001). In addition, stretching modifies the morphology, cellular orienta-
tion, and shape of fibroblasts (mechanical response), with the upregulation of genes
encoding for cellular and extracellular components (β1 integrins, β-actin, and types
I and III collagen) (biochemical response), described through the process of mecha-
notransduction (Kaneko et al., 2009). With the position of the cell maintained within
the tissue architecture by external complexes (integrins, costameres), which act to
set a “baseline” level of cell tension (tensegrity), changes in this tension result in the
cell’s response to a mechanical load (Wu, Fannin, Rice, Wang, & Blough, 2011).
The cell increases its stress state by contracting and applying both a mechanical
load onto itself, with a subsequent biochemical response, directed to adjacent cells,
in direct contact or through the extracellular matrix (Banes et al., 1995). This is a
critical response designed to accommodate and acclimate functional loading in tis-
sues (Orr et al., 2006). With mechanical force being a primary regulator of biologi-
cal functions, mediating development of tissue (i.e. skeletal muscle, tendons etc.),
and influencing diverse cellular processes (cell growth, differentiation, protein syn-
thesis) (Alenghat & Ingber, 2002; Ingber, 2003; Wu et al., 2011), the propagation of
a biochemical signal associated with stretching (Koh, Petersen, Pizza, & Brooks,
2003b; Pizza et al., 2002) alludes to its possible mechanoregulatory role. With the
response of fibroblasts to mechanical load being dependent upon stretching magni-
tude, frequency, and duration (Wang, Thampatty, Lin, & Im, 2007; Wang, Yang, Li,
& Shen, 2004), the loading of tissue based on stretching intensity (low, medium,
high), through the process of mechanotransduction, may shed light on how this
mechanical stimuli may influence the muscle and connective tissue.
Stretching and Inflammatory Cells
The expression of neutrophils relative to lengthening and isometric contractions and

passive stretching was investigated in a study wherein 71 adult male mice were
divided into lengthening and isometric contractions or passive stretching groups
(Pizza et al., 2002). During lengthening, the extensor digitorum longus was stimu-
lated and lengthened to 20% of its optimal fibre length, held at its optimal length
(isometric), or lengthened without stimulation (passive stretching). Each exercise
consisted of 75 repetitions lasting 5 min, with controls allowed to roam freely. Mice
were sacrificed at 6 h or 3 days after initial in situ procedure, with immunohisto-
chemistry for both neutrophils and macrophages conducted on 10 μm cross sections
excised from the midbelly of the extensor digitorum longus. Inflammatory cells
were counted and expressed as a number per cubic millimetre, with the number of
fibres invaded by these cells counted and expressed as a percentage of the total
number. At 3 days, passive stretching and isometric contractions accounted for a
5.5- and 3.7-fold increase in neutrophils, respectively, with lengthening contrac-
tions accounting for a 7.9-fold increase, compared to control. This rise in neutro-
phils occurred whether muscle activity resulted in injury or not, suggesting that
passive stretching was a stimulus for one or more chemoattractants for neutrophils,
without any overt impairments in function or histological disruption (Pizza et al.,
2002). This result needs to be investigated further, for it may be related to the dis-
turbed homeostasis and dysregulation of intracellular Ca2+ which is associated with
the proteolytic activation of the ubiquitously expressed calpain expression in skel-
etal muscle, responsible for degrading contractile and other muscle proteins
(Fatouros & Jamurtas, 2016; Tu, Levin, Hamilton, & Borodinsky, 2016). In addi-
tion, passive stretching was observed to promote protection of adult mice muscle
following lengthening contractions, reducing the force deficit, the number of overtly
injured fibres, and the accumulation of inflammatory cells (Koh et al., 2003a; Koh
& Brooks, 2001).
Using complimentary approaches, an ex vivo model consisting of excised mouse
subcutaneous tissue, and an in vivo model consisting of a subcutaneous microsurgi-
cal injury to the back of mice, a study investigated the effects of a brief tissue stretch
on TGF-β1, and connective tissue matrix remodelling (Bouffard et al., 2008).
Elevated extracellular levels of TGF-β1 is associated with the activation of fibro-
blasts, a major ECM effector, responsible for the increased synthesis of collagen,
elastin, and proteoglycan (substrates of the ECM) (Bouffard et al., 2008). With
changing levels of mechanical forces (i.e. immobilisation, exercise, and stretching)
implicated in connective tissue remodelling (Bouffard et al., 2008), externally
applied mechanical forces are believed to reduce collagen deposition during tissue
repair and scar formation (Cummings & Tillman, 1992). In the ex vivo model, sub-
cutaneous tissue, kept in organ culture for 4 days, was stretched (20% strain for
10 min, 1 day post excision) or not, with mice in the in vivo model randomised into
a stretching (20–30% strain for 10 min twice a day for 7 days) or non-stretching
group. The stretching protocol involved stretching of the trunk of the mouse while
Stretching and Inflammatory Cells 93
suspended by its tail, forcing it to extend its front and hind limbs towards a surface
slightly inclined relative to vertical, with the distance measured, being the differ-
ence between the ipsilateral hip and shoulder joint during stretching and at rest
(~20–30% greater during stretching). TGF-β1 protein was lower in the stretched
tissue (ex vivo), with a significant rise in type 1 procollagen (in vivo) in the absence
of stretch, suggesting that brief tissue stretching attenuates increases in soluble
TGF-β1 and type 1 procollagen. These results hint that stretching is relevant in
response to tissue injury (Bouffard et al., 2008).
A study by Smith et al. documented whether stretching (i.e. static or ballistic
stretching) was responsible for DOMS (Smith et al., 1993). Most studies investigat-
ing DOMS and stretching were concerned whether stretching (i.e. static, passive,
active, ballistic, dynamic, and PNF) can influence DOMS (Lund, Vestergaard-
Poulsen, Kanstrup, & Sejrsen, 1998; Wessel & Wan, 1994). Based on eligible ran-
domised clinical trials, a Cochrane Collaboration Review investigated whether
stretching before, after, or before and after exercise was beneficial in treating or
preventing DOMS (Herbert, De Noronha, & Kamper, 2011). This review concluded
that regardless of when the muscle was stretched, no clinically important reductions
in DOMS occur (Herbert et al., 2011). However, stretching intensity was not taken
into account.
The primary outcome measured in the Smith et al. (1993) study, in response to
static or ballistic stretching of a similar intensity and duration, was creatine kinase.
Participants randomly assigned to either stretching group performed three identical
sets of 17 stretching exercises, with the static group remaining stationary during
each 60 s stretch and the ballistic group bouncing in time to a metronome (60
bounces/min). Blood samples were collected at pre- and 24, 48, 72, 96, and 120 h
postexercise for creatine kinase, with rate perceived exertion scores recorded fol-
lowing each stretch using the Borg 6–20 scale. Although significant increases in
DOMS were observed in both groups, static stretching was associated with more.
Unfortunately, no mention was made of the stretching intensity (i.e. mild, discom-
fort, pain). Interestingly, the time course and extent of DOMS associated with static
stretching is similar to that reported for eccentric exercise, with discomfort ensuing
in the first 24–48 h, peaking between 24 and 72 h, and subsiding within 5–7 days
(Ebbeling & Clarkson, 1989).
With stretching associated with morphological changes to muscle fibre (i.e. dis-
ruption of sarcomere) (Gomes et al., 2007), the disruption in the homeostasis of
Ca2+ (Armstrong et al., 1993), increases in neutrophils (Koh et al., 2003a; Pizza
et al., 2002), and a primary cause of DOMS (Smith et al., 1993), interest in this
manuscript was concerned with stretching intensity. With stretching defined as a
force (intensity) responsible for stressing connective and muscle tissue (Martins
et al., 2013), the magnitude of force identified with stretching intensity (low,
medium, high) may be responsible for stimulating a response of the interconnected
layers from the macro (muscles and tendons) to the micro level (sarcomeres). In
other words, the tissues and cells of each layer may respond differently to low- (i.e.
no pain, gentle) versus high-intensity (i.e. pain, discomfort) stretching, conceivably
responsible for inducing, increasing, maintaining, or alleviating the acute inflammatory
response. The activation and mobilisation of immune cells mediated by the

cytokines, released by the injured muscle, triggers inflammation (Fatouros &
Jamurtas, 2016). To examine this assumption, three studies were designed. To our
knowledge, studies one and two were the first to investigate stretching in humans
with reference to blood biomarkers for inflammation and the acute inflammatory
response. The first study investigated whether high-intensity passive static stretch-
ing (IS) (i.e. discomfort with some pain), used in many sports, in particular in aes-
thetic (i.e. gymnastics) and martial arts (i.e. taekwondo, judo, etc.), is responsible
for an acute inflammatory response. High-sensitivity C-reactive protein and pro-
inflammatory cytokines (IL-1β, TNF-α, and IL-6) were measured. The second study
compared various passive static stretching intensities (low, medium, and high) to
determine if a relationship exists between these different intensities and the expres-
sion of the acute inflammatory response. The blood biomarker measured was
hsCRP. With study three, the applicability of passive static stretching intensity (low
and high) in relation to recovery of the musculoskeletal tissue from an unaccus-
tomed eccentric exercise was investigated. Unaccustomed eccentric exercise has
been credited with the development of DOMS and strength loss (Fatouros &
Jamurtas, 2016). Although perceived muscle soreness and muscle function are indi-
rect measures, the totality of an injury to the muscle is a decrease in its ability to
develop maximum force (Faulkner et al., 1993). DOMS is a form of acute inflam-
mation, with the sensation of perceived muscle soreness representing inflammatory
pain (Lieber & Friden, 1993; MacIntyre et al., 1995; Smith, 1991). Soreness and
pain are considered contributing factors in regulating muscle activity, with muscular
strength remaining depressed throughout the period of soreness (Talag, 1973). This
interruption to the muscle tissue is associated with a disorganisation of the myofi-
brillar material, in particular, a focal disruption of the sarcomeres, with the Z-disc
being the most vulnerable structure to an eccentric exercise-induced injury (Friden
& Lieber, 2001). By specifically focusing on stretching intensity, what is presented
is the concept that the magnitude of stretching intensity may be a contributing factor
responsible for aiding in the proper recovery of the muscle. This has implications
for exercise training and athletic performance, and may shorten the time of rehabili-
tation from musculoskeletal disorders and pain.
References
Abdel-Aziem, A. A., Draz, A. H., Mosaad, D. M., & Abdelraou, O. R. (2013). Effect of body posi-
tion and type of stretching on hamstring flexibility. International Journal of Medical Research
and Health Sciences, 2, 399–406.
Ackermann, M. A., Ziman, A. P., Strong, J., Zhang, Y., Hartford, A. K., Ward, C. W., et al. (2011).
Integrity of the network sarcoplasmic reticulum in skeletal muscle requires small ankyrin 1.
JCS, 124, 3619–3630.
ACSM. (2006). ACSM’s guidelines for exercise testing and prescription. Baltimore, MD: Lippincot
Williams Wilkins.
References 95
Adams, R. J., & Schwartz, A. (1980). Comparative mechanisms for contraction of cardiac and
skeletal muscle. Chest, 78, S123–S139.
Adelstein, R. S. (1983). Regulation of contractile proteins by phosphorylation. JCI, 72, 1863–1866.
Agarkova, I., Ehler, E., Lange, S., Schoenuer, R., & Perriard, J. C. (2003). M-band: A safeguard for
sarcomere stability. Journal of Muscle Research and Cell Motility, 24, 191–203.
Agarkova, I., & Perriard, J. C. (2005). The M-band: An elastic web that crosslinks thick filaments
in the center of the sarcomere. Trends in Cell Biology, 15, 477–485.
Agbulut, O., Li, Z., Perie, S., Ludosky, M.-A., Paulin, D., Cartaud, J., et al. (2001). Lack of desmin
results in abortive muscle regeneration and modifications in synaptic structure. Cell Motility
and the Cytoskeleton, 49, 51–66.
Ahearne, M. (2014). Introduction to cell-hydrogel mechanosensing. Interface Focus, 4, 1–12.
Ahn, A. C., Tewari, M., Poon, C.-S., & Phillips, R. S. (2006). The limits of reductionism in medi-
cine: Could systems biology offer an alternative? PLoS Medicine, 3, 709–713.
Akerstrom, T. C. A., Steensberg, A., Keller, P., Keller, C., Penkowa, M., & Pedersen, B. K. (2005).
Exercise induces interleukin-8 expression in human skeletal muscle. The Journal of Physiology,
563, 507–516.
Akhmanova, M., Osidak, E., Domogatsky, S., Rodin, S., & Domogatskaya, A. (2015). Physical,
spatial, and molecular aspects of extracellular matrix of in vivo niches and artificial scaffolds
relevant to stem cells research. Stem Cells International, 2015, 1–35.
Akimoto, T., Furudate, M., Saitoh, M., Sugiura, K., Waku, T., Akama, T., et al. (2002). Increased
plasma concentrations of intercellular adhesion molecule-1 after strenuous exercise associated
with muscle damage. European Journal of Applied Physiology, 86, 185–190.
Akira, S., Hirano, T., Taga, T., & Kishimoto, T. (1990). Biology of multifunctional cytokines: IL-6
and related molecules (IL-1 and TNF). FASEB, 4, 2860–2867.
Akira, S., Taga, T., & Kishimoto, T. (1993). Interleukin-6 in biology and medicine. Advances in
Immunology, 54, 1–78.
Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology
of the Cell (4th ed.). New York: Garland Science.
Alenghat, F. J., & Ingber, D. E. (2002). Mechanotransduction: All signals point to cytoskeleton,
matrix, and integrins. Science’s STKE, 119, 1–4.
Alexander, R. M. (1991). Energy-saving mechanisms in walking and running. The Journal of
Experimental Biology, 160, 55–69.
Alexander, R. M., & Bennett-Clark, H. C. (1977). Storage of elastic strain energy in muscle and
other tissues. Nature, 265, 114–117.
Allen, D. G. (2001). Eccentric muscle damage: Mechanisms of early reduction of force. Acta
Physiologica Scandinavica, 171, 311–319.
Al-Mokdad, M., Shibata, F., & Nakagawa, H. (1997). Effects of cytokine-induced neutrophil che-
moattractants (CINCs) on shape change, adhesiveness and phagocytosis of rat neutrophils.
Biological & Pharmaceutical Bulletin, 20, 920–923.
Anastasi, G., Amato, A., Tarone, G., Vita, G., Monici, M. C., Magaudda, L., et al. (2003).
Distribution and localization of vinculin-talin-integrin system and dystrophin-glycoprotein
complex in human skeletal muscle - immunohistochemical study using confocal laser scanning
microscopy. Cells, Tissues, Organs, 175, 151–164.
Anastasi, G., Cutroneo, G., Gaeta, R., Di Mauro, D., Arco, A., Consolo, A., et al. (2009).
Dystrophin-glycoprotein complex and vinculin-talin-integrin system in human adult cardiac
muscle. International Journal of Molecular Medicine, 23, 149–159.
Anastasi, G., Cutroneo, G., Rizzo, G., Arco, A., Santoro, A., Bramanti, P., et al. (2004). Sarcoglycan
and integrin localization in normal human skeletal muscle: A confocal laser scanning micro-
scope study. European Journal of Histochemistry, 48, 245–252.
Anastasi, G., Cutroneo, G., Santoro, A., Arco, A., Rizzo, G., Bramanti, P., et al. (2008). Costameric
proteins in human skeletal muscle during muscular inactivity. Journal of Anatomy, 213,
284–295.
Anastasi, G., Cutroneo, G., Santoro, G., Arco, A., Rizzo, G., Trommino, C., et al. (2006). Integrins,
muscle agrin and sarcoglycans during muscular inactivity conditions: An immunohistochemi-
cal study. European Journal of Histochemistry, 50, 327–336.
Anderson, J., Li, Z., & Goubel, F. (2001). Passive stiffness is increased in soleus muscle of desmin
knockout mouse. Muscle & Nerve, 24, 1090–1092.
evance of stretch intensity and position - A systematic review. Frontiers in Psychology, 6, 1128.
Archambault, J., Tsuzaki, M., Heerzog, W., & Banes, A. J. (2002). Stretch and interleukin-1β
induce matrix metalloproteinases in rabbit tendon cells in vitro. Journal of Orthopaedic
Research, 20, 36–39.
Arey, B. J. (Ed.). (2012). The role of glycosylation in receptor signaling. London: InTech.
Armstrong, R. B. (1990). Initial events in exercise-induced muscular injury. Medicine and Science
in Sports and Exercise, 22, 429–435.
Armstrong, R. B., Duan, C., Delp, M. D., Hayes, D. A., Glen, G. M., & Allen, G. D. (1993).
Elevations in rat soleus muscle [Ca2+] with passive stretch. Journal of Applied Physiology, 74,
2990–2997.
Armstrong, R. B., Ogilvie, R. W., & Schwane, J. A. (1983). Eccentric exercise-induced injury to
rat skeletal muscle. Journal of Applied Physiology: Respiratory, Environmental and Exercise
Physiology, 54, 80–93.
Arnold, L., Henry, A., Poron, F., Baba-Amer, Y., Van Rooijen, N., Plonquet, A., et al. (2007).
Inflammatory monocytes recruited after skeletal muscle injury switch into antiinflammatory
macrophages to support myogenesis. JEM, 204, 1057–1069.
Aronson, D., Wojtaszewski, J. F., Thorell, A., Nygren, J., Zangen, D., Richter, E. A., et al. (1998).
Extracellular-regulated protein kinase cascades are activated in response to injury in human
skeletal muscle. American Journal of Physiology. Cell Physiology, 275, C555–C561.
Ashcroft, G. S. (1999). Bidirectional regulation of macrophage function by TGF-β. Microbes and
Infection, 1, 1275–1282.
Atabai, K., Jame, S., Azhar, N., Kuo, A., Lam, M., Mckleory, W., et al. (2009). Mfge8 diminishes
the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macro-
phages. The Journal of Clinical Investigation, 119, 3713–3722.
Atherton, P., Stutchbury, B., Jethwa, D., & Ballestrem, C. (2016). Mechanosensitive components
of integrin adhesions: Role of vinculin. Experimental Cell Research, 343, 21–27.
Atherton, P., Stutchbury, B., Wang, D.-Y., Jethwa, D., Tsang, R., Meiler-Rodriguez, E., et al. (2015).
Vinculin controls talin engagement with the actomyosin machinery. Nature Communications,
6, 10038.
Atreya, R., Mudter, J., Finotto, S., Mullberg, J., Jostock, T., Wirtz, S., et al. (2000). Blockade of
interleukin trans signaling T-cell resistance against apoptosis in chronic intestinal inflamma-
tion: Evidence in crohn disease and experimental colitis in vivo. Nature Medicine, 6, 583–588.
Aumailley, M., & Smyth, N. (1998). The role of laminins in basement membrane function. Journal
of Anatomy, 193, 1–21.
Avela, J., Kyrolainen, H., & Komi, P. (1999). Altered reflex sensitivity after repeated and pro-
longed passive muscle stretching. Journal of Applied Physiology, 86, 1283–1291.
Ayalon, G., Hostettler, J. D., Hoffman, J., Kizhatil, K., Davis, J. Q., & Bennett, V. (2011).
Ankyrin-B interactions with spectrin and dynactin-4 are required for dystrophin-based protec-
tion of skeletal muscle from exercise injury. JBC, 286, 7370–7378.
Badylak, S. F. (2002). The extracellular matrix as a scaffold for tissue reconstruction. Seminars in
Cell & Developmental Biology, 13, 377–383.
Baldwin, A. K., Simpson, A., Steer, R., Cain, S. A., & Kielty, C. M. (2013). Elastic fibres in health
and disease. Expert Reviews in Molecular Medicine, 15, 1–30.
Balnave, C. D., & Allen, D. G. (1995). Intracellular calcium and force in single mouse muscle
fibres following repeated contractions with stretch. The Journal of Physiology, 488, 25–36.
Balnave, C. D., Davey, D. F., & Allen, D. G. (1997). Distribution of sarcomere length and intra-
cellular calcium in mouse skeletal muscle following stretch-induced injury. The Journal of
References 97
Bandy, W., Irion, J. M., & Briggler, M. (1997). The effect of time and frequency of static stretching
on flexibility of the hamstring muscles. Physical Therapy, 77, 1090–1096.
Banes, A. J., Tsuzaki, M., Yamamoto, J., Fischer, T., Brigman, B., Brown, T., et al. (1995).
Mechanotransduction at the cellular level: The detection, interpretation, and diversity of
responses to mechanical signals. Biochemistry and Cell Biology, 73, 349–365.
Bang, M.-L., Centner, T., Fornoff, F., Geach, A. J., Gothardt, M., Mcnabb, M., et al. (2001). The
complete gene sequence of titin, expression of an unusual 700-kDa titin isoform, and its inter-
action with obscurin identify a novel Z-line to I-band linking system. Circulation Research,
89, 1065–1072.
Bang, M.-L., Li, X., Littlefield, R., Bremner, S., Thor, A., Knowlton, K. U., et al. (2006). Nebulin-
deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal
muscle. JCB, 173, 905–916.
Bao, Z. Z., Lakonishok, M., Kaufman, S. J., & Horwitz, A. F. (1993). α7β1 integrin is a component
of the myotendinous junction on skeletal muscle. JCS, 106, 579–590.
Barash, I. A., Mathew, L., Lahey, M., Greaser, M. L., & Lieber, R. L. (2005). Muscle LIM protein
plays both structural and functional roles in skeletal muscle. American Journal of Physiology-
Cell Physiology, 289, C1312–C1320.
Barbieri, E., & Sestili, P. (2012). Reactive oxygen species in skeletal muscle signaling. Journal of
Signal Transduction, 2012, 1–17.
Barczyk, M., Carracedo, S., & Gulberg, D. (2010). Integrins. Cell and Tissue Research, 339,
269–280.
Baron, L., & Wynn, T. A. (2011). Fibrosis is regulated by Th2 and Th17 responses and by
dynamic interactions between fibroblasts and macrophages. American Journal of Physiology.
Gastrointestinal and Liver Physiology, 300, G723–G728.
Barral, J. M., & Epstein, H. F. (1999). Protein machines and self assembly in muscle organization.
BioEssays, 21, 813–823.
Barral, J. M., Hutagalung, A. H., Brinker, A., Hartl, U., & Epstein, H. F. (2002). Role of the myosin
assembly protein UNC-45 as a molecular chaperone for myosin. Science, 295, 669–671.
Barton, B. E. (1997). IL-6: Insights into novel biological activities. Clinical Immunology and
Immunopathology, 85, 16–20.
Barua, B., Winkelmann, D. A., White, H. D., & Hitchcock-Degregori, S. E. (2012). Regulation
of actin-myosin interaction by conserved periodic sites of tropomyosin. Proceedings of the
National Academy of Sciences of the USA, 109, 18425–18430.
Batters, C., Veigel, C., Homsher, E., & Sellers, J. R. (2014). To understand muscle you must take
it apart. Frontiers in Physiology, 5, 1–14.
Baumann, H., & Gauldie, J. (1990). Regulation of acute phase plasma protein genes by hepatocyte
stimulating factors and other mediators of inflammation. Molecular Biology & Medicine, 7,
147–159.
Beer, S. (1975). Preface. In H. R. Maturana & F. J. Varela (Eds.), Autopoietic systems. BLC Report
9. Champaign, IL: University of Illinois.
Behm, D. G., & Chaouachi, A. (2011). A review of the acute effects of static and dynamic stretch-
ing on performance. European Journal of Applied Physiology, 11, 2633–2651.
Behm, D. G., & Kibele, A. (2007). Effects of differing intensities of static stretching on jump per-
formance. European Journal of Applied Physiology, 101, 587–594.
Belcastro, A., Arthur, G., Albisser, T., & Raj, D. (1996). Heart, liver and skeletal muscle myeloper-
oxidase activity during exercise. Journal of Applied Physiology, 80, 1331–1335.
Belcastro, A., Shewchuk, L. D., & Raj, D. A. (1998). Exercise-induced muscle injury: A calpain
hypothesis. Molecular and Cellular Biochemistry, 179, 135–145.
Belcastro, A. N., Maclean, I., & Gilchrist, J. S. (1985). Biochemical basis of muscle fatigue associ-
ated with repetitious contractions of skeletal muscle. The International Journal of Biochemistry,
17, 447–453.
Belcastro, A. N., Parkhouse, W., Dobson, G., & Gilchrist, J. S. (1988). Influence of exercise on
cardiac and skeletal muscle myofibrillar proteins. Molecular and Cellular Biochemistry, 83,
27–36.
Belkin, A. M., Zhidkova, N. I., Balzac, F., Altruda, F., Tomatis, D., Maier, A., et al. (1996). β1D
integrin displaces the β1A isoform in striated muscles: Localization at junctional structures and
signaling potential in nonmuscle cells. JCB, 132, 211–226.
Bellin, R. M., Huiatt, T. W., Critchley, D. R., & Robson, R. M. (2001). Synemin may function to
directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres.
The Journal of Biological Chemistry, 276, 32330–32337.
Bencze, M., Negroni, E., Vallese, D., Yacoub-Youssef, H., Chaouch, S., Wolff, A., et al. (2012).
Proinflammatory macrophages enhance the regenerative capacity of human myoblasts by mod-
ifying their kinetics of proliferation and differentiation. Molecular Therapy, 20, 2168–2179.
Benjamin, M., Evans, E. J., & Copp, L. (1986). The histology of tendon attachments to bone in
man. Journal of Anatomy, 149, 89–100.
Benjamin, M., & Ralphs, J. R. (1998). Fibrocartilage in tendons and ligaments-an adaptation to
compressive load. Journal of Anatomy, 193, 481–494.
Berchtold, M. W., Brinkmeier, H., & Muntener, M. (2000). Calcium ion in skeletal muscle: Its
crucial role for muscle function, plasticity, and disease. Physiological Reviews, 80, 1216–1265.
Bershadsky, A. D., Balaban, N. Q., & Geiger, B. (2003). Adhesion-dependent cell mechanosensi-
tivity. Annual Review of Cell and Developmental Biology, 19, 677–695.
Bevilacqua, M. P., Pober, J. S., Mendrick, D. L., Cotran, R. S., & Gimbrone Jr., M. A. (1987).
Identification of an inducible endothelial-leukocyte adhesion molecule. Proceedings of the
Bickel, M. (1993). The role of interleukin-8 in inflammation and mechanisms of regulation.
Journal of Periodontology, 64, 456–460.
Biebricher, C. K., Nicolis, G., & Schuster, P. (1995). Self-organization in the physico-chemical and
life sciences. Brussels, Belgium: European Commission.
Bissell, M. J., & Aggeler, J. (1987). Dynamic reciprocity: How do extracellular matrix hormones
direct gene expression? Progress in Clinical and Biological Research, 249, 251–262.
Bo Lauersen, J., Bertelsen, D. M., & Bo Andersen, L. (2014). The effectiveness of exercise inter-
ventions to prevent sports injuries: A systematic review and meta-analysis of randomised con-
trolled trials. British Journal of Sports Medicine, 48, 871–877.
Bobbert, M. F., Ettema, G. C., & Huijing, P. A. (1990). The force-length relationship of a muscle-
tendon complex: Experimental results and model calculations. European Journal of Applied
Border, W. A., & Noble, N. A. (1994). Transforming growth factor beta in tissue fibrosis. The New
England Journal of Medicine, 331, 1286–1292.
Borejdo, J., Ushakov, D. S., & Akopova, I. (2002). Regulatory and essential light chains of myosin
rotate equally during contraction of skeletal muscle. Biophysical Journal, 82, 3150–3159.
Borovikov, Y. S., & Gusev, N. B. (1983). Effect of troponin-tropomyosin complex and Ca2+ on
conformational changes. European Journal of Biochemistry, 136, 363–369.
Bosurgi, L., Manfredi, A. A., & Rovere-Querini, P. (2011). Macrophages in injured skeletal mus-
cle: A perpetuum mobile causing and limiting fibrosis, prompting or restricting resolution and
regeneration. Frontiers in Immunology, 2, 1–10.
Bouffard, N. A., Cutroneo, K. R., Badger, G. J., White, S. L., Buttoplph, T. R., Paul Ehrlich, H.,
et al. (2008). Tissue stretch decreases soluble TGF-β1 and Type-1 procollagen in mouse sub-
cutaneous connective tissue: Evidence from ex vivo and in vivo models. Journal of Cellular
Bowen, T., Jenkins, R. H., & Fraser, D. J. (2013). MicroRNAs, transforming growth factor beta-1,
and tissue fibrosis. The Journal of Pathology, 229, 274–285.
Bowman, W. (1840). On the minute structure and movements of voluntary muscle. Philosophical
Transactions of the Royal Society B, 130, 457–501.
Bozyczko, D., Decker, C., Muschler, J., & Horwitz, A. F. (1989). Integrin on developing and adult
skeletal muscle. Experimental Cell Research, 183, 72–91.
Bradley, P. S., Olsen, P. D., & Portas, M. D. (2007). The effect of static, ballistic and proprioceptive
neuromuscular facilitation stretching on vertical jump performance. Journal of Strength and
Conditioning Research, 21, 223–226.
References 99
Brand, P. (1984). Clinical mechanics of the hand. St. Louis, MO: CV Mosby.
Brandenburg, J. P. (2006). Duration of stretch does not influence the degree of force loss following
static stretching. The Journal of Sports Medicine and Physical Fitness, 46, 526–534.
Brigitte, M., Schilte, C., Plonquet, A., Baba-Amer, Y., Henri, A., Charlier, C., et al. (2010). Muscle
resident macrophages control the immune cell reaction in a mouse model of notexin-induced
myoinjury. Arthritis and Rheumatism, 62, 268–279.
Brockett, C. L., Morgan, D. L., & Proske, U. (2001). Human hamstring muscles adapt to eccentric
exercise by changing optimum length. Medicine & Science in Sports & Exercise, 33, 783–790.
Brooks, S. V., Zerba, E., & Faulkner, J. A. (1995). Injury to muscle fibres after single stretches of
passive and maximally stimulated muscles in mice. The Journal of Physiology, 488, 459–469.
Brown, D. L., Kao, W. W. Y., & Greenhalgh, D. G. (1992). Apoptosis down-regulates inflamma-
tion under the advancing epithelial wound edge: Delayed patterns in diabetes and improvement
with topical growth factors. Surgery, 121, 327–380.
Brown, L., & Hill, L. (1991). Some observations on variations in filament overlap in tetanized
muscle fibres and fibres stretched during tetanus, detected in the electron microscope after
rapid fixation. Journal of Muscle Research and Cell Motility, 12, 171–182.
Bruton, A. (2002). Muscle plasticity response to training and detraining. Physiotherapy, 88,
398–408.
Bruunsgaard, H., Galbo, H., Halkjaer, K. J., Johansen, T. L., Maclean, D. A., & Pedersen, B. K.
(1997). Exercise-induced increase in serum interleukin-6 in humans is related to muscle dam-
age. The Journal of Physiology, 499, 833–841.
Bruunsgaard, H., Skinhoj, P., Qvist, J., & Pedersen, B. K. (1999). Elderly humans show prolonged
in vivo inflammatory activity during pneumococcal infections. The Journal of Infectious
Diseases, 180, 551–554.
Burkholder, T. J. (2007). Mechanotransduction in skeletal muscle. Frontiers in Bioscience, 12,
174–191.
Burkholder, T. J., Fingado, S., Baron, S., & Lieber, R. L. (1994). Relationship between muscle
fiber types and sizes and muscle architectural properties in the mouse hind limb. Journal of
Morphology, 221, 177–190.
Burkin, D. J., & Kaufman, S. J. (1999). The α7β1 integrin in muscle development and disease. Cell
and Tissue Research, 296, 183–190.
Burridge, K., & Guilluy, C. (2016). Focal adhesions, stress fibers and mechanical tension.
Experimental Cell Research, 343, 14–20.
Butterfield, T. A., Best, T. M., & Merrick, M. A. (2006). The dual roles of neutrophils and macro-
phages in inflammation: A critical balance between tissue damage and repair. JAT, 41, 457–465.
Bystrom, J., Evans, I., Newson, J., Stables, M., Toor, I., Van Rooijen, N., et al. (2008). Resolution-
phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP.
Blood, 112, 4117–4127.
Caldwell, J. E., Heiss, S. G., Mermall, V., & Cooper, J. A. (1989). Effects of CapZ, an actin cap-
ping protein of muscle, on the polymerization of actin. The Biochemist, 28, 8506–8514.
Campbell, I. D., & Humphries, M. J. (2011). Integrin structure, activation, and interactions. Cold
Spring Harbor Perspectives in Biology, 2011, 1–14.
Campbell, K. P. (1995). Three muscular dystrophies: Loss of cytoskeleton-extracellular matrix
linkage. Cell, 80, 675–679.
Camus, G., Pincemail, J., Ledent, M., Juchmes-Ferr, A., Lamy, M., Deby-Duupont, G., et al.
(1992). Plasma levels of polymorphonuclear elastase and myeloperoxidase after uphill walk-
ing and downhill running at similar energy cost. International Journal of Sports Medicine, 13,
443–446.
Cannon, J. G., & Kluger, M. J. (1983). Endogenous pyrogen activity in human plasma after exer-
cise. Science, 220, 617–619.
Cannon, J. G., & St. Pierre, B. A. (1998). Cytokines in exertion-induced skeletal muscle injury.
Molecular and Cellular Biochemistry, 179, 159–167.
Canon, J., Meydani, S., Fielding, R. A., Fiatarone, M. A., Meydani, M., Farhangmehr, M., et al.
(1991). Acute phase response in exercise II. Associations between vitamin E, cytokines, and
muscle proteolysis. American Journal of Physiology. Regulatory, Integrative and Comparative

Physiology, 260, R1235–R1240.
Capetanaki, Y., Bloch, R. J., Kouloumenta, A., Mavroidis, M., & Psarras, S. (2007). Muscle inter-
mediate filaments and their links to membranes and membranous organelles. Experimental
Cell Research, 313, 2063–2076.
Caplan, N., Rogers, R., Parr, M., & Hayes, P. (2009). The effect of proprioceptive neuromus-
cular facilitation and static stretch training on running mechanics. Journal of Strength and
Carlos, T. M., & Harlan, J. M. (1994). Leukocyte-endothelial adhesion molecules. Blood, 84,
2068–2101.
Carlson, B. M., & Faulkner, J. A. (1983). The regeneration of skeletal muscle fibers following
injury: A review. Medicine and Science in Sports and Exercise, 15, 187–198.
Carver, W., & Goldsmith, E. C. (2013). Regulation of tissue fibrosis by the biomechanical environ-
ment. BioMed Research International, 2013, 1–10.
Castell, J. V., Gomez-Lechion, M. J., David, M., Fabra, R., Trullenque, R., & Heinrich, P. C.
(1990). Acute-phase response of human hepatocytes: Regulation of acute-phase protein syn-
thesis by interleukin-6. Hepatology, 12, 1179–1186.
Cavaillon, J. M. (2001). Pro-versus anti-inflammatory cytokines: Myth or reality. Cellular and
Molecular Biology (Noisy-le-Grand, France), 47, 695–702.
Chaar, V., Romana, M., Tripetter, J., Broquere, C., Huisse, M.-G., Hue, O., et al. (2011). Effect of
strenuous physical exercise on circulating cell-derived microparticles. Clinical Hemorheology
and Microcirculation, 47, 15–25.
Chalmers, G. (2004). Re-examination of the possible role of golgi tendon organ and muscle spin-
dle reflexes in proprioceptive facilitation muscle stretching. Sports Biomechanics, 3, 159–183.
Chan, M. H., Carey, A. L., Watt, M. J., & Febbraio, M. A. (2004). Cytokine gene expression in
human skeletal muscle during concentric contraction: Evidence that IL-8, like IL-6, is influ-
enced by glycogen availability. American Journal of Physiology. Regulatory, Integrative and
Comparative Physiology, 287, R322–R327.
Chandler, T., Kinbler, W., Uhl, T., Wooten, B., Kiser, A., & Stone, E. (1990). Flexibility compari-
sons of junior elite tennis players to other athletes. The American Journal of Sports Medicine,
18, 134–136.
Chapman, M. A., Meza, R., & Lieber, R. L. (2016). Skeletal muscle fibroblasts in health and dis-
ease. Differentiation, 92, 108–115.
Charge, S. B., & Rudnicki, M. A. (2004). Cellular and molecular regulation of muscle regenera-
tion. Physiological Reviews, 84, 209–238.
Charturvedi, V., Dye, D. E., Kinnear, B. F., Van Kuppevelt, T. H., Grounds, M. D., & Coombe,
D. R. (2015). Interactions between skeletal muscle myoblast and the extracellular matrix
revealed by a serum free culture system. PLoS One, 2015, 1–27.
Charvet, B., Ruggiero, F., & Le Guellec, D. (2012). The development of the myotendinous junc-
tion. A review. Muscles, Ligaments, and Tendons Journal, 2, 53–63.
Chen, C. S. (2008). Mechanotransduction - A field pulling together? Journal of Cell Science, 121,
3285–3292.
Chen, C. S., Tan, J., & Tien, J. (2004). Mechanotransduction at cell-matrix and cell-cell contacts.
Annual Review of Biomedical Engineering, 6, 275–302.
Chen, Q., Fisher, D. T., Clancy, K. A., Gauquet, J. M., Wang, W. C., Unger, E., et al. (2006). Fever-
range thermal stress promotes lymphocyte trafficking across high endothelial venules via an
interleukin 6 trans-signaling mechanism. Nature Immunology, 7, 1299–1308.
Cheung, K., Hume, P., & Maxwell, L. (2003). Delayed onset muscle soreness: Treatment strategies
and performance factors. Sports Medicine, 33, 145–164.
Chew, M. W. K., & Squire, J. M. (1995). Packing of a-helical coiled-coil myosin rod in vertebrate
muscle thick filaments. Journal of Structural Biology, 115, 233–249.
Chikenji, T., Gingery, A., Zhao, C., Vanhees, M., Moriya, T., Reisdorf, R., et al. (2014).
Transforming growth factor-β (TGF-β) expression is increased in the subsynovial connective
tissue in a rabbit model of carpal tunnel syndrome. PLoS One, 9, 1–6.
References 101
Chiquet, M. (1999). Regulation of extracellular matrix gene expression by mechanical stress.

Matrix Biology, 18, 417–426.
Chiquet, M., Renedo, A. S., Huber, F., & Fluck, M. (2003). How do fibroblasts translate mechani-
cal signals into changes in extracellular matrix production. Matrix Biology, 22, 73–80.
Ciobanasu, C., Faivre, B., & Le Clainche, C. (2013). Integrating actin dynamics, mechanotrans-
duction and integrin activation: The multiple functions of actin binding proteins in focal adhe-
sions. European Journal of Cell Biology, 92, 339–348.
Ciobanasu, C., Faivre, B., & Le Clainche, C. (2014). Actomyosin-dependent formation of the
mechanosensitive talin-vinculin complex reinforces actin anchoring. Nature Communications,
5, 3095.
Clark, K., Langeslag, M., Figdor, C. G., & Van Leeuwen, F. N. (2007). Myosin II and mechano-
transduction: A balancing act. Trends in Cell Biology, 17, 178–186.
Clark, K. A., Mcelhinny, A. S., Beckerle, M. C., & Gregorio, C. C. (2002). Striated muscle cyto-
architecture: An intricate web of form and function. Annual Review of Cell and Developmental
Biology, 18, 637–706.
Clarkson, P. M., & Hubal, M. J. (2002). Exercise-induced muscle damage in humans. American
Journal of Physical Medicine & Rehabilitation, 81, S52–S69.
Coffey, D. S. (1998). Self organization, complexity and chaos: The new biology of medicine.
Nature Medicine, 4, 882–885.
Colditz, I., Zwahlen, R., Dewald, B., & Baggiolini, M. (1989). In vivo inflammatory activity of
neutrophil-activating factor, a novel chemotactic peptide derived from human monocytes. The
American Journal of Pathology, 134, 755–760.
Condon, S. M., & Hutton, R. S. (1987). Soleus muscle electromyographic activity and ankle dorsi-
flexion range of motion during four stretching procedures. Physical Therapy, 67, 24–30.
Connelly, R., & Back, A. (1998). Mathematics and tensegrity: Group and representation theory
make it possible to form a complete catalogue of “strut-cable” constructions with prescribed
symmetries. American Scientist, 86, 142–151.
Conover, G. M., & Gregorio, C. C. (2011). The desmin coil 1B mutation K190 impairs nebulin
Z-disc assembly and destabilizes actin thin filaments. JCS, 124, 3464–3476.
Cornwell, A., Nelson, A. G., & Sidaway, B. (2002). Acute effects of stretching on the neurome-
chanical properties of the triceps surae complex. European Journal of Applied Physiology, 86,
428–434.
Costa, M. L., Escaleira, R., Cataldo, A., Oliveira, F., & Mermelstein, C. S. (2004). Desmin:
Molecular interactions and putative functions of the muscle intermediate filament protein.
Brazilian Journal of Medical and Biological Research, 37, 1819–1830.
Couchman, J. R. (2003). Syndecans: Proteoglycan regulators of cell-surface microdomains. Nature
Reviews. Molecular Cell Biology, 4, 926–937.
Craig, R. W., & Padron, R. (2004). Molecular structure of the sarcomere. In A. C. Engel &
C. Franzini-Armstrong (Eds.), Myology (3rd ed.). New York: McGraw-Hill.
Craig, S. W., & Pardo, J. V. (1983). Gamma actin, spectrin, and intermediate filament proteins colo-
calize with vinculin at costameres, myofibril-to-sarcolemma attachment sites. Cell Motility, 3,
449–462.
Crick, F. H. C. (1953). The packing of a-helices: Simple coiled-coils. Acta Cryst, 6, 689–697.
Critchley, D. R. (2005). Genetic, biochemical and structural approaches to talin function.
Biochemical Society Transactions, 33, 1308–1312.
Cummings, G. S., & Tillman, L. J. (Eds.). (1992). Remodeling of dense connective tissue in normal
adult tissues. Philadelphia: F.A. Davis.
Damas, P., Ledoux, D., Nys, M., Vrindts, Y., De Groote, D., Franchimont, P., et al. (1992). Cytokine
serum level during severe sepsis in human IL-6 as a marker of severity. Annals of Surgery, 215,
356–362.
Daniels, K., & Solursh, M. (1991). Modulation of chondrogenesis by the skeleton and extracellular
matrix. Journal of Cell Science, 100, 249–254.
Davidson, C. J., Ganion, L. R., Gehlsen, G. M., Bverhoestra, B., Roepke, J. E., & Sevier, T. L.
(1997). Rat tendon morphologic and functional changes resulting from soft tissue mobilization.
Medicine and Science in Sports and Exercise, 29, 313–319.
Dayton, W. R., Reville, W. J., Goll, D. E., & Stromer, M. H. (1976). A Ca2+-activated protease
possibly involved in myofibrillar protein turnover. Partial characterization of the purified
enzyme. The Biochemist, 15, 2159–2167.
de Almeida Ribeiro Jr., E., Pinotsis, N., Ghisleni, A., Salmazo, A., Konarev, P. V., Kostan, J., et al.
(2014). The structure and regulation of human muscle α-actinin. Cell, 159, 1447–1460.
De Palma, L., Marinelli, M., Pavan, M., & Bertoni-Freddari, C. (2011). Involvement of the muscle-
tendon junction in skeletal muscle atrophy: An ultrastructural study. Romanian Journal of
Morphology and Embryology, 52, 105–109.
Decary, S., Mouly, V., Hamida, C. B., Sautet, A., Barbet, J. P., & Butler-Browne, G. S. (1997).
Replicative potential and telomere length in human skeletal muscle: Implications for satellite
cell-mediated gene therapy. Human Gene Therapy, 8, 1429–1438.
DeDeyne, P. G. (2001). Application of passive stretch and its implications for muscle fibers.
Physical Therapy, 81, 819–827.
Dejong, L. D., Nieuwboer, A., & Aufdemkampe, G. (2007). The hemiplegic arm: Interrater reli-
ability and concurrent validity of passive range of motion measurements. Journal of Disability
and Rehabilitation, 29, 1442–1448.
del Rio, A., Perez-Jimenez, R., Liu, R., Roca-Cusachs, P., Fernandez, J. M., & Sheetz, M. P. (2009).
Stretching single talin rod molecules activates vinculin binding. Science, 323, 638–641.
Detmers, P. A., Powell, D. E., Walz, A., Clark-Lewis, I., Baggiolini, M., & Cohn, Z. A. (1991).
Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte
adhesion and phagocytosis. Journal of Immunology, 147, 4211–4217.
Discher, D. E., Janmey, P. A., & Wang, Y.-L. (2005). Tissue cells feel and respond to the stiffness
of their substrate. Science, 310, 1139–1143.
Discher, D. E., Mooney, D. J., & Zandstra, P. W. (2009). Growth factors, matrices, and forces
combine and control stem cells. Science, 324, 1673–1677.
Drenth, J. P., Van Hum, S. H., Van Deuren, M., Pesman, G. J., Van Der Ven-Jongekrijg, J., et al.
(1995). Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo Tnf-
alpha and IL-1 beta production. Journal of Applied Physiology, 79, 1497–1503.
Dubravec, D. B., Spriggs, D. R., Mannick, J. A., & Rodrick, M. L. (1990). Circulation human
peripheral blood granulocytes synthesize and secrete tumor necrosis factor α. Proceedings of
the National Academy of Sciences of the USA, 87, 6758–6761.
Duclos, T. W. (2000). Function of C-reactive protein. Annals of Medicine, 32, 274–278.
Duenwald, S. E., Vanderby Jr., R., & Lakes, R. S. (2009). Viscoelastic relaxation and recovery of
tendon. Annals of Biomedical Engineering, 37, 1131–1140.
Dufort, C. C., Paszek, M. J., & Weaver, V. M. (2011). Balancing forces: Architectural control of
mechanotransduction. Nature Reviews. Molecular Cell Biology, 12, 308–319.
Dunne, J. L., Ballantyne, C. M., Beaudet, A. L., & Ley, K. (2002). Control of leukocyte rolling
velocity in TNF-α-induced inflammation by LFA-1 and Mac-1. Blood, 99, 336–341.
Duque, G. A., & Descoteaux, A. (2014). Macrophage cytokines: Involvement in immunity and
infectious diseases. Frontiers in Immunology, 5, 1–12.
Ebbeling, C. B., & Clarkson, P. M. (1989). Exercise-induced muscle damage and adaptation.
Egebald, M., Rasch, M. G., & Weaver, V. M. (2010). Dynamic interplay between the collagen scaf-
fold and tumor evolution. Current Opinion in Cell Biology, 22, 697–706.
Elliot, B. (2006). Biomechanics and tennis. British Journal of Sports Medicine, 40, 392–396.
Elmore, S. (2007). Apoptosis: A review of programmed cell death. Toxicologic Pathology, 35,
495–516.
Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (2006). Matrix elasticity directs stem cell
lineage application. Cell, 126, 677–689.
Ervasti, J. (2003). Costameres: The Achilles’ heel of Herculean muscle. The Journal of Biological
Chemistry, 278, 13591–13594.
Ervasti, J. (2007). Dystrophin, its interactions with other proteins, and implications for muscular
dystrophy. Biochemica et Biophysica Acta, 1772, 108–117.
References 103
Ervasti, J., & Campbell, K. P. (1993). A role for the dystrophin-glycoprotein complex as a trans-
membrane linker between laminin and actin. JCB, 122, 809–823.
Etnyre, B. R., & Abraham, L. D. (1986). Gains in range of ankle dorsiflexion using three popular
stretching techniques. American Journal of Physical Medicine, 65, 189–196.
Etnyre, B. R., & Lee, E. J. (1987). Comments on proprioceptive neuromuscular facilitation stretch-
ing techniques. Research Quarterly for Exercise and Sport, 58, 184–188.
Evans, R. R., Robson, R. M., & Stromer, M. H. (1984). Properties of smooth muscle vinculin. The
Journal of Biological Chemistry, 259, 3916–3924.
Evans, W. J., & Cannon, J. G. (1991). The metabolic effects of exercise-induced muscle damage.
Exercise and Sport Sciences Reviews, 19, 99–125.
Eyckmans, J., Boudou, T., Yu, X., & Chen, C. S. (2011). A hitchhiker’s guide to mechanobiology.
Developmental Cell, 21, 35–47.
Fadok, V. A., Bratton, D. L., Konowal, A., Freed, P. W., Wescott, J. Y., & Henson, P. M. (1989).
Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine pro-
duction through autocrine/paracrine mechanisms involving TGF-beta, PGE2 and PAF. JCI,
101, 890–898.
Fatouros, I., & Jamurtas, A. Z. (2016). Insights into the molecular etiology of exercise-induced
inflammation: Opportunities for optimizing performance. Journal of Inflammation Research,
9, 175–186.
Faulkner, G., Lanfranchi, G., & Valle, G. (2001). Telethonin and other new proteins of the Z-disc
of skeletal muscle. IUBMB Life, 51, 275–282.
Faulkner, G., Pallavicini, A., Comelli, A., Salamon, M., Bortoletto, G., Ievolella, C., et al. (2000).
FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z-disc of skeletal muscle. The
Journal of Biological Chemistry, 275, 41234–41242.
Faulkner, R. A., Brooks, S. V., & Opiteck, J. A. (1993). Injury to skeletal muscle fibers during
contractions: Conditions of occurrence and prevention. Physical Therapy, 73, 911–921.
Feasson, L., Stockholm, D., Freyssenet, D., Richard, I., Duguez, S., Beckmann, J. S., et al. (2002).
Molecular adaptations of neuromuscular disease-associated proteins in response to eccentric
exercise in human skeletal muscle. The Journal of Physiology, 543, 297–306.
Febbraio, M. A., Hiscock, N., Sacchetti, M., Fischer, C. P., & Pedersen, B. K. (2004). Interleukin-6
is a novel factor mediating glucose homeostasis during skeletal muscle contraction. Diabetes,
53, 1643–1648.
Febbraio, M. A., & Pedersen, B. K. (2002). Muscle-derived interleukin-6: Mechanisms for activa-
tion and possible biological roles. FASEB, 16, 1335–1347.
Febbraio, M. A., & Pedersen, B. K. (2005). Contraction-induced myokine production and release:
Is skeletal muscle an endocrine organ? Exercise and Sport Sciences Reviews, 33, 114–119.
Feland, J. B., Myrer, J. W., & Merrill, R. M. (2001). Acute changes in hamstring flexibility: PNF
versus static stretch in senior athletes. Physical Therapy in Sport, 2, 186–193.
Feland, J. B., Myrer, J. W., Schulthies, S., Fellingham, G., & Measom, G. (2001). The effect of
duration of stretching of the hamstring muscle group for increasing range of motion in people
aged 65 years or older. Physical Therapy, 81, 1110–1117.
Fernando, M. R., Reyes, J. L., Iannuzzi, J., Leung, G., & Mckay, D. M. (2014). The pro-
inflammatory cytokine, interleukin-6, enhances the polarization of alternatively activated mac-
rophages. PLoS One, 9, 1–12.
Field, C. J., Gougeon, R., & Marliss, E. B. (1991). Circulating mononuclear cell numbers and
function during intense exercise and recovery. Journal of Applied Physiology, 71, 1089–1097.
Fielding, R. A., Manfredi, T. J., Ding, W., Fiatarone, M. A., Evans, W. J., & Cannon, J. G. (1993).
Acute phase response in exercise III. Neutrophil and IL-1β accumulation in skeletal muscle.
American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 265,
R166–R172.
Figarella-Branger, D., Civatte, M., Bartoli, C., & Pellissier, J. F. (2003). Cytokines, chemokines,
and cell adhesion molecules in inflammatory myopathies. Muscle & Nerve, 28, 659–682.
Flynn, J. L., Chan, J., & Lin, P. L. (2011). Macrophages and control of granulomatous inflamma-
tion in tuberculosis. Mucosal Immunology, 4, 271–278.
Fox, J. E., Goll, D. E., Reynolds, C. C., & Phillips, D. R. (1985). Identification of two proteins
(actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease
during platelet aggregation. The Journal of Biological Chemistry, 260, 1060–1066.
Frangos, J. A., Mcintire, L. V., & Eskin, S. G. (1988). Shear stress induced stimulation of
mammalian cell metabolism. Biotechnology and Bioengineering, 32, 1053–1060.
Frank, D., Kuhn, C., Katus, H. A., & Frey, N. (2006). The sarcomeric Z-disc: A nodal point in
signalling and disease. Journal of Molecular Medicine, 84, 446–468.
Franzini-Armstrong, C. (Ed.). (1994). The sarcoplasmic reticulum and the transverse tubules.
New York: McGraw-Hill.
Freitas, S. R., Mendes, B., Le Sant, G., Andrade, R. J., Nordez, A., & Milanovic, Z. (2017). Can
chronic stretching changes the muscle-tendon mechanical properties? A review. Scandinavian
Journal of Medicine & Science in Sports, 28, 794–806.
Frenette, J., St-Pierre, M., Cote, C. H., Mylona, E., & Pizza, F. X. (2002). Muscle impairment
occurs rapidly and precedes inflammatory cell accumulation after mechanical loading.
American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, 282,
R351–R357.
Freytes, D. O., Wan, L. Q., & Vunjak-Novakovic, G. (2009). Geometry and force control of cell
function. Journal of Cellular Biochemistry, 108, 1047–1058.
Friden, J., Kjorell, U., & Thornell, L.-E. (1984). Delayed muscle soreness and cytoskeletal altera-
tions: An immunocytological study in man. International Journal of Sports Medicine, 5, 15–18.
Friden, J., & Lieber, R. L. (2001). Eccentric exercise-induced injuries to contractile and cytoskel-
etal muscle fibre components. Acta Physiologica Scandinavica, 171, 321–326.
Friden, J., Sjostrom, M., & Ekblom, B. (1981). A morphological study of delayed muscle soreness.
Experientia, 37, 506–507.
Friden, J., Sjostrom, M., & Ekblom, B. (1983). Myofibrillar damage following intense eccentric
exercise in man. International Journal of Sports Medicine, 4, 170–176.
Frost, H. M. (1990). Skeletal structural adaptations to mechanical usage (SATMU): 4. Mechanical
influences on intact fibrous tissues. The Anatomical Record, 226, 433–439.
Frost, R. A., & Lang, C. H. (2005). Skeletal muscle cytokines: Regulation by pathogen-associated
molecules and catabolic hormones. Current Opinion in Clinical Nutrition and Metabolic Care,
8, 255–263.
Fukunaga, T., Kawakami, Y., Kuno, S., Funato, K., & Fukashiro, S. (1997). Muscle architecture
and function in humans. Journal of Biomechanics, 30, 457–463.
Fukunaga, T., Roy, R. R., Shellock, F. G., Hodgson, J. A., Lee, P. L., Kwong-Fu, H., et al. (1992).
Physiological cross-sectional area of human leg muscles based on magnetic resonance imagin-
ing. Journal of Orthopaedic Research, 10, 926–934.
Fuller, G. M., & Grenett, H. E. (1989). The structure and function of the mouse hepatocyte stimu-
lating factor. Annals of the New York Academy of Sciences, 557, 31–44.
Funatsu, T., Higuchi, H., & Ishiwata, S. (1990). Elastic filaments in skeletal muscle revealed by
selective removal of thin filaments with gelsolin. JCB, 110, 53–62.
Funk, D. C., Swank, A. M., Mikla, B. M., Fagan, T. A., & Farr, B. K. (2003). Impact of prior exer-
cise on hamstring flexibility: A comparison of proprioceptive neuromuscular facilitation and
static stretching. National Strength & Conditioning Association, 17, 489–492.
Furst, D. O., Osborn, M., Nave, R., & Weber, K. (1988). The organization of titin filaments in the
half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: A map of
ten nonrepetitive epitopes starting at the Z line extends close to the M line. The Journal of Cell
Biology, 106, 1563–1572.
Furze, R. C., & Rankin, S. M. (2008). Neutrophil mobilization and clearance in the bone marrow.
Gabay, C. (2006). Interleukin-6 and chronic inflammation. Arthritis Research & Therapy, 8, S3–S8.
Gabay, C., & Kushner, I. (1999). Acute-phase proteins and other systemic responses to inflamma-
tion. The New England Journal of Medicine, 340, 448–454.
References 105
Gabriel, H., Schwartz, L., Steffens, G., & Kindermann, W. (1992). Immunoregulatory hormones,
circulating leukocyte and lymphocyte subpopulations before and after endurance exercise of
different intensities. International Journal of Sports Medicine, 1992, 359–366.
Gajdosik, R. L. (2001). Passive extensibility of skeletal muscle: Review of literature with clinical
implications. Clinical Biomechanics (Bristol, Avon), 16, 87–101.
Gajdosik, R. L., & Bohannon, R. W. (1987). Clinical measurement of range of motion-review of
goniometry emphasizing reliability and validity. Physical Therapy, 67, 1867–1872.
Galbraith, C. G., Yamada, K. M., & Sheetz, M. P. (2002). The relationship between force and focal
complex development. JCB, 159, 695–705.
Gans, C. (Ed.). (1991). Efficiency, effectiveness, perfection, optimization. Their use in understand-
ing vertebrate evolution. Oxford, UK: Oxford Univ Press.
Gans, C., & Abbot, S. G. (1991). Muscle architecture in relation to function. Journal of
Biomechanics, 24, 53–65.
Garrett, W. E., Nikolaou, P. K., Rtibbeck, B. M., Glisson, R. R., & Seaber, A. (1988). The effect
of muscle architecture on the biomechanical failure properties of skeletal muscle under passive
extension. American Journal of Sports Medicine, 16, 7–11.
Gattazzo, F., Urciuolo, A., & Bonaldo, P. (2014). Extracellular matrix: A dynamic microenviron-
ment for stem cell niche. Biochimica et Biophysica Acta, 1840, 2506–2519.
Gautel, M. (1996). The super-repeats of titin/connectin and their interactions: Glimpses at sarco-
meric assembly. Advances in Biophysics, 33, 27–37.
Gautel, M. (2011). The sarcomeric cytoskeleton: Who picks up the strain? Current Opinion in Cell
Biology, 23, 39–46.
Gautel, M., Goulding, D., Bullard, B., Weber, K., & Furst, D. O. (1996). The central Z-disk region
of titin is assembled from a novel repeat in variable copy numbers. Journal of Cell Science,
109, 2747–2754.
Gautel, M., Mues, A., & Young, P. (1999). Control of sarcomeric assembly: The flow of informa-
tion on titin. Reviews of Physiology, Biochemistry and Pharmacology, 138, 97–137.
Geiger, B., & Bershadsky, A. (2002). Exploring the neighbourhood: Adhesion-coupled cell mecha-
nosensors. Cell, 110, 139–142.
Geiger, B., Spatz, J. P., & Bershadsky, A. D. (2009). Environmental sensing through focal adhe-
sions. Nature Reviews. Molecular Cell Biology, 10, 21–33.
Gelberman, R., Goldberg, V., & An, K.-N. (1988). Tendon. Park Ridge, IL: American Academy of
Orthopaedic Surgeons.
Gillies, A. R., & Lieber, R. L. (2011). Structure and function of the skeletal muscle extracellular
matrix. Muscle & Nerve, 44, 318–331.
Gleeson, M. (2000). Interleukins and exercise. The Journal of Physiology, 529, 1–1.
Goldberg, A. L., Etlingger, J. D., Goldspink, D. F., & Jablecki, C. (1975). Mechanism of work-
induced hypertrophy of skeletal muscle. Medicine and Science in Sports and Exercise, 7,
248–261.
Goldmann, W. H. (2012). Mechanotransduction and focal adhesions. Cell Biology International,
36, 649–652.
Goldspink, D. (1977). The influence of immobilization and stretch on protein turnover of rat skel-
etal muscle. The Journal of Physiology, 264, 267–282.
Goldspink, G. (1999). Changes in muscle mass and phenotype and the expression of autocrine and
systemic growth factors by muscle in response to stretch and overload. Journal of Anatomy,
194, 323–334.
Goldspink, G. (2005). Mechanical signals, IGF-I gene slicing, and muscle adaptation. Physiology,
20, 232–238.
Goldstein, M. A., Schroeter, J. P., & Michael, L. H. (1991). Role of the Z-band in the mechanical
properties of the heart. The FASEB Journal, 5, 2167–2174.
Goldstein, M. A., Schroeter, J. P., & Sass, R. L. (1982). The Z-band lattice in a slow skeletal
muscle. Journal of Muscle Research and Cell Motility, 3, 333–348.
Goll, D. E., Dayton, W. R., Singh, I., & Robson, R. M. (1991). Studies of the alpha-actinin/actin
interaction in the Z-disk by using calpain. JBC, 266, 8501–8510.
Goll, D. E., Thompson, V. F., Li, H., Wei, W., & Cong, J. (2003). The calpain system. Physiological
Reviews, 83, 731–801.
Gomes, A. R. S., Cornachione, A., Salvini, T. F., & Mattiello-Sverzut, A. C. (2007). Morphological
effects of two protocols of passive stretch over the immobilized rat soleus muscle. Journal of
Anatomy, 210, 328–335.
Gontier, Y., Taivainen, A., Fontao, L., Sonnenberg, A., Van Der Flier, A., Carpen, O., et al. (2005).
The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the
sarcolemma via muscle-specific filamins. JCS, 118, 3739–3749.
Gordon, A. M., Honmsher, E., & Regnier, M. (2000). Regulation of contraction in striated muscle.
Physiological Reviews, 80, 853–924.
Gordon, S., & Taylor, P. R. (2005). Monocyte and macrophage heterogeneity. Nature Reviews
Goulam Houssen, Y., Gusachenko, I., Schanne-Klein, M.-C., & Allain, J.-M. (2011). Monitoring
micrometer-scale collagen organization in rat-tail tendon upon mechanical strain using second
harmonic microscopy. Journal of Biomechanics, 44, 2047–2052.
Granger, D. N., & Senchenkova, E. (2010). Inflammation and the microcirculation. San Rafael,
CA: Morgan & Claypool Life Sciences.
Granzier, H., & Labeit, S. (2005). Titin and its associated proteins: The third myofilament system
of the sarcomere. Advances in Protein Chemistry, 71, 89–119.
Granzier, H., & Labeit, S. (2006). The giant muscle protein titin is an adjustable molecular spring.
Exercise and Sport Sciences Reviews, 34, 50–53.
Granzier, H. L., Akster, H. A., & Ter Keurs, H. E. (1991). Effect of thin filament length on the
force-sarcomere length relation of skeletal muscle. American Journal of Physiology. Cell
Physiology, 260, C1060–C1070.
Granzier, H. L., & Labeit, S. (2004). The giant protein titin: A major player in myocardial mechan-
ics, signaling and disease. Circulation Research, 94, 284–295.
Greaser, M. (2001). Identification of new repeating motifs in titin. Proteins, 43, 145–149.
Green, C. L., Brown, L., Stewart, J. J., Xu, Y., Litwin, V., & Mc Closkey, T. W. (2011).
Recommendations for the validation of flow cytometric testing during drug development: I
instrumentation. Journal of Immunological Methods, 363, 104–119.
Green, L. J., Mould, P., & Humphries, M. J. (1998). The integrin β subunit. The International
Journal of Biochemistry & Cell Biology, 30, 179–184.
Greenhalgh, D. G. (1998). The role of apoptosis in wound healing. The International Journal of
Biochemistry & Cell Biology, 30, 1019–1030.
Gregorio, C. C., Trombitas, K., Centner, T., Kolmerer, B., Stier, G., Kunke, K., et al. (1998). The
NH2 terminus of titin spans the Z-disc: Its interaction with a novel 19-kD ligand (T-cap) is
required for sarcomeric integrity. JCB, 143, 1013–1027.
Grounds, M. D. (1987). Phagocytosis of necrotic muscle in muscle isografts is influenced by the
strain, age, and sex of host mice. The Journal of Pathology, 153, 71–82.
Grounds, M. D., Sorokin, L., & White, J. (2005). Strength and the extracellular matrix-muscle
interface. Scandinavian Journal of Medicine & Science in Sports, 15, 381–391.
Gruys, E., Toussaint, M. J. M., Niewold, T. A., & Koopmans, S. J. (2005). Acute phase reaction
and acute phase proteins. Journal of Zhejiang University. Science. B, 6, 1045–1056.
Guharay, F., & Sachs, F. (1984). Stretch-activated single ion channel currents in tissue-cultured
embryonic chick skeletal muscle. The Journal of Physiology, 352, 685–701.
Guissard, N., & Duchateau, J. (2006). Neural aspects of muscle stretching. Exercise and Sport
Sciences Reviews, 34, 154–158.
Gumerson, J. D., & Michele, D. E. (2011). The dystrophin-glycoprotein complex in the prevention
of muscle damage. Journal of Biomedicine & Biotechnology, 2011, 1–13.
Hack, A. A., Groh, M. E., & Mcnally, E. M. (2000). Sarcoglycans in muscular dystrophy.
Microscopy Research and Technique, 48, 167–180.
References 107
Hack, E., De Groot, E. R., Felt-Bersma, R. J. F., Nuijjens, J. H., Strack Van Schijndel, R. J. M.,
Eerenberg-Belmer, A. J. M., et al. (1989). Increased plasma levels of interleukin-6 in sepsis.
Blood, 74, 1704–1710.
Haining, A. W. M., Lieberthal, T. J., & Del Rio Hernandez, A. (2016). Talin: A mechanosensitive
molecule in health and disease. The FASEB Journal, 30, 2073–2085.
Hamill, O. (1997). Special topic: Molecular mechanisms of mechanotransduction. Annual Review
of Physiology, 59, 573–574.
Hamill, O. P., & Martinac, B. (2001). Molecular basis of mechanotransduction in living cells.
Physiological Reviews, 81, 685–740.
Handel, M., Horstmann, T., Dickhuth, H., & Gulch, R. (1997). Effects of contract-relax stretch-
ing training on muscle performance in athletes. European Journal of Applied Physiology, 76,
400–408.
Hannan, M. T., & Freeman, J. (1977). The population of ecology organizations. American Journal
of Sociology, 82, 929–964.
Hashizume, M., & Mihara, M. (2011). The roles of interleukin-6 in the pathogenesis of rheumatoid
arthritis. Arthritis, 2011, 1–8.
Haslett, C. (1992). Resolution of acute inflammation and the role of apoptosis in the tissue fate of
granulocytes. Clinical Science, 83, 639–648.
Heinrich, P. C., Castell, J. V., & Andus, T. (1990). Interleukin-6 in the acute phase response.
Biochemical Journal, 265, 621–625.
Heinrich, P. C., Horn, F., Graeve, L., Dittrich, E., Kerr, I., Muller-Newen, G., et al. (1998).
Interleukin-6 and related cytokines: Effect on the acute phase reaction. Zeitschrift für
Ernährungswissenschaft, 37, 43–49.
Helge, J. W., Stallknecht, B., Pedersen, B. K., Galbo, H., Kiens, B., & Richter, E. A. (2003). The
effect of graded exercise on IL-6 release and glucose uptake in human skeletal muscle. The
Journal of Physiology, 546, 299–305.
Hellsten, Y., Frandsen, U., Orthenblad, N., & Sjodin, B. (1997). Xanthine oxidase in human skel-
etal muscle following eccentric exercise: A role in inflammation. The Journal of Physiology,
498, 239–248.
Henics, T., & Wheatley, D. N. (1999). Cytoplasmic vacuolation, adaptation and cell death: A view
on new perspectives and features. Biology of the Cell, 91, 485–498.
Hensriksson-Larsen, K., Wretling, M., Lorentzen, R., & Oberg, L. (1992). Do muscle fiber size
and fiber angulation correlate in pennated human muscles? European Journal of Applied
Herbert, R. D., De Noronha, M., & Kamper, S. J. (2011). Stretching to prevent or reduce muscle
soreness after exercise. Cochrane Database of Systematic Reviews, 7, 1–47.
High, D. M., Howley, E. T., & Franks, B. D. (1989). The effects of static stretching and warm-up
on prevention of delayed onset muscle soreness. Research Quarterly for Exercise and Sport,
60, 357–361.
Higuchi, H., Yoshioka, T., & Maruyama, K. (1988). Positioning of actin filaments and tension gen-
eration in skinned muscle fibres released after stretch beyond overlap of the actin and myosin
filaments. Journal of Muscle Research and Cell Motility, 9, 491–498.
Hindle, K. B., Whitcomb, T. J., Briggs, W. O., & Hong, J. (2012). Proprioceptive neuromuscu-
lar facilitation (PNF): Its mechanisms and effects on range of motion and muscular function.
Journal of Human Kinetics, 31, 105–113.
Hirani, N., Antonicelli, F., Strieter, R. M., Wiesener, M. S., & Ratcliffe, P. J. (2001). The regulation
of interleukin-8 by hypoxia in human macrophages-a potential role in the pathogenesis of the
acute respiratory distress syndrome (ARDS). Molecular Medicine, 7, 685–697.
Hiscock, N., Chan, M. H. S., Bisucci, T., Darby, I. A., & Febbraio, M. A. (2004). Skeletal myo-
cytes are a source of interleukin-6 mRNA expression and protein release during contraction:
Evidence of fiber type specificity. FASEB, 18, 992–994.
Hoffman, B. D., Grashoff, C., & Schwartz, M. A. (2011). Dynamic molecular processes mediate
cellular mechanotransduction. Nature, 475, 316–323.
Holmberg, J., & Durbeej, M. (2013). Laminin-211 iun skeletal muscle function. Cell Adhesion &
Migration, 7, 111–121.
Honda, H., Kimura, H., & Rostami, A. (1990). Demonstration and phenotype characterization of
resident macrophages in rat skeletal muscle. Immunology, 70, 272–277.
Horowits, R. (1992). Passive force generation and titin isoforms in mammalian skeletal muscle.
Biophysical Journal, 61, 392–398.
Horowits, R., Kempner, E. S., Bisher, M. E., & Podolsky, R. J. (1986). A physiological role for titin
and nebulin. Nature, 323, 160–164.
Horowits, R., Luo, G., Zhang, J. Q., & Herrera, A. H. (1996). Nebulin and nebulin-related proteins
in striated muscle. Advances in Biophysics, 33, 143–150.
Horowits, R., & Podolsky, R. J. (1987). The positional stability of thick filaments in activated skel-
etal muscle depends on sarcomere length: Evidence for the role of titin filaments. The Journal
of Cell Biology, 105, 2217–2223.
Huang, H., Kamm, R. D., & Lee, R. T. (2004). Cell mechanics and mechanotransduction: Pathways,
probes, and physiology. American Journal of Physiology. Cell Physiology, 287, C1–C11.
Huang, J., & Forsberg, N. E. (1998). Role of calpain in skeletal muscle protein degradation.
Proceedings of the National Academy of Sciences of the USA, 95, 12100–12105.
Huang, S., Sultan, C., & Ingber, D. E. (2006). Tensegrity, dynamic networks, and complex systems
biology: Emergence in structural and information networks within living cells. Boston, MA:
Springer.
Huijbregts, P. A. (2001). Muscle injury, regeneration, and repair. The Journal of Manual &
Manipulative Therapy, 9, 9–16.
Huijing, P. A., & Baan, G. C. (2001). Myofascial force transmission causes interaction between
adjacent muscles and connective tissue: Effects of blunt dissection and compartmental fas-
ciotomy on length force characteristics of rat extensor digitorum longus muscle. Archives of
Physiology and Biochemistry, 109, 97–109.
Hultborn, H., Illert, M., & Santini, M. (1976). Convergence on interneurones mediating the recip-
rocal Ia inhibition of motoneurones. Acta Physiologica Scandinavica, 96, 368–391.
Humphrey, J. D., Dufresne, E. R., & Schwartz, M. A. (2014). Mechanotransduction and extracel-
lular matrix homeostasis. Nature Reviews. Molecular Cell Biology, 15, 802–812.
Humphries, J. D., Wang, P., Streuli, C., Geiger, B., Humphries, M. J., & Ballestrem, C. (2007).
Vinculin controls focal adhesion formation by direct interactions with talin and actin. JCB,
179, 1043–1057.
Hutagalung, A. H., Landsverk, M. L., Price, M. G., & Epstein, H. F. (2002). The UCS family of
myosin chaperones. Journal of Cell Science, 115, 3983–3990.
Huxley, H. E. (1969). The mechanism of muscular contraction. Science, 164, 1356–1366.
Huxley, H. E., & Hanson, J. (1954). Changes in the cross-striations of muscle during contraction
and stretch and their structural interpretations. Nature, 173, 973–976.
Hynes, R. O. (1992). Integrins: Versatility, modulation and signaling in cell adhesion. Cell, 69,
11–25.
Hynes, R. O. (2009). The extracellular matrix: Not just pretty fibrils. Science, 326, 1216–1219.
Hynes, R. O., & Naba, A. (2012). Overview of the matrisome-an inventory of extracellular matrix
constituents and functions. Cold Spring Harbor Perspectives in Biology, 2012, 1–16.
Ingber, D. E. (1997). Tensegrity: The architectural basis of cellular mechanotransduction. Annual
Review of Physiology, 59, 575–599.
Ingber, D. E. (1998). The architecture of life. Scientific American, 278, 48–57.
Ingber, D. E. (2003). Mechanobiology and diseases of mechanotransduction. Annals of Medicine,
8, 564–577.
Ingber, D. E. (2004). The mechanochemical basis of cell and tissue regulation. Molecular and
Cellular Biology, 1, 53–68.
Ingber, D. E. (2006). Cellular mechanotransduction: Putting all the pieces together again. FASEB,
20, 811–827.
References 109
Ingber, D. E. (2008a). Tensegrity-based mechanosensing from macro to micro. Progress in

Biophysics and Molecular Biology, 97, 163–179.
Ingber, D. E. (2008b). Tensegrity and mechanotransduction. Journal of Bodywork and Movement
Therapies, 12, 198–200.
Ingber, D. E., Dike, I., Hansen, I., Karp, S., Liley, H., Maniotis, A., et al. (1994). Cellular tenseg-
rity: Exploring how mechanical changes in the cytoskeleton regulate cell growth, migration,
and tissue pattern during morphogenesis. International Review of Cytology, 150, 173–224.
Italiani, P., & Boraschi, D. (2014). From monocytes to M1/M2 macrophages: Phenotypical vs
functional differentiation. Frontiers in Immunology, 5, 1–22.
Itsumi, M., Yoshikai, Y., & Yamada, H. (2009). IL-15 is critical for the maintenance and innate
functions of self-specific CD8+ T cells. European Journal of Immunology, 38, 1784–1793.
Jackson, D., Jarret, H., Bailey, D. M., Kausek, J., Swanson, J., & Powell, J. (1978). Injury predic-
tion in the young athlete: A preliminary report. The American Journal of Sports Medicine, 6,
6–14.
Jacobs, C. A., & Sciacia, A. D. (2011). Factors that influence the efficacy of stretching programs
for patients with hypomobility. Sports Health, 3, 520–523.
Jain, S., Gautam, V., & Naseem, S. (2011). Acute-phase proteins: As diagnostic tool. Journal of
Pharmacy & Bioallied Sciences, 3, 118–127.
Jalali, S., Del Pozo, M. A., Chen, K.-D., Miao, H., Li, Y.-S., Shyy, J. Y., et al. (2001). Integrin-
mediated mechanotransduction requires its dynamic interaction with specific extracellular
matrix (ECM) ligands. Proceedings of the National Academy of Sciences of the USA, 98,
1042–1046.
Jamney, P. A., & Mcculloch, C. A. (2007). Cell mechanics: Integrating cell responses to mechani-
cal stimuli. Annual Review of Biomedical Engineering, 9, 1–34.
Jarvinen, T. A. H., Jarvinen, T. L. N., Kaariainen, M., Kalimo, H., & Jarvinen, M. (2005). Muscle
injuries: Biology and treatment. The American Journal of Sports Medicine, 33, 745–764.
Jones, D. A., Newham, D. J., Round, J. M., & Tolfree, S. E. J. (1986). Experimental human mus-
cle damage: Morphological changes in relation to other indices of damage. The Journal of
Jonsdottir, I. H., Schjerling, P., Ostrowski, K., Asp, S., Richter, E. A., & Pedersen, B. K. (2000).
Muscle contractions induces interleukin-6 mRNA production in rat skeletal muscles. The
Kabat, H. (1950). Studies on neuromuscular dysfunction: XIII. New concepts and techniques of
neuromuscular re-education for paralysis. Permanente Foundation Medical Bulletin, 8, 21–143.
Kad, N. M., Kim, S., Warshaw, D. M., Vanburren, P., & Baker, J. E. (2005). Single-myosin cross-
bridge interactions with actin filaments regulated by troponin-tropomyosin. Proceedings of the
Kanda, K., Sugama, K., Hayashida, H., Sakuma, J., Kawakami, Y., Miura, S., et al. (2013).
Eccentric exercise-induced delayed-onset muscle soreness and changes in markers of muscle
damage and inflammation. Exercise Immunology Review, 19, 72–85.
Kaneko, D., Sasazaki, Y., Kikuchi, T., Ono, T., Nemoto, K., Matsumoto, H., et al. (2009). Temporal
effects of cyclic stretching on distribution and gene expression of integrin and cytoskeleton by
ligament fibroblasts in vitro. Connective Tissue Research, 50, 263–269.
Kaplanski, G., Marin, V., Montenero-Julian, F., Mantovani, A., & Farnarier, C. (2003). IL-6: A reg-
ulator of the transition from neutrophil to monocyte recruitment during inflammation. Trends
in Immunology, 24, 25–29.
Karalaki, M., Filli, S., Philippou, A., & Koutsilieris, M. (2009). Muscle regeneration: Cellular and
molecular events. in vivo (Vol. 23, pp. 779–796).
Kastelic, J., Galeski, A., & Baer, E. (1978). Multicomposite structure of tendon. Connective Tissue
Research, 6, 11–23.
Katzemich, A., Kreiskother, N., Alexandrovich, A., Elliot, C., Schock, F., & Bullard, B. (2012).
The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the
flight muscle of Drosophila. JCS, 125, 3367–3379.
Kawakami, Y., Abe, T., & Fukunaga, T. (1993). Muscle-fiber pennation angles are greater in hyper-
trophied than in normal muscles. Journal of Applied Physiology, 74, 2740–2744.
Kawakami, Y., Abe, T., Kuno, S., & Fukunaga, T. (1995). Training-induced changes in muscle
architecture and specific tension. European Journal of Applied Physiology, 72, 37–43.
Keller, C., Steensberg, A., Pilegaard, H., Osada, T., Saltin, B., Pedersen, B. K., et al. (2001).
Transcriptional activation of the IL-6 gene in human contracting skeletal muscle: Influence of
muscle glycogen content. FASEB, 15, 2748–2750.
Kenny, P. A., Liston, E. M., & Higgins, D. G. (1999). Molecular evolution of immunoglobulin and
fibronectin domains in titin and related muscle proteins. Gene, 232, 11–23.
Khan, M. M. (2008). Immunopharmacology. New York: Springer.
Khan, S., & Sheetz, M. (1997). Force effects on biochemical kinetics. Annual Review of
Biochemistry, 66, 785–805.
Kilicarslan, A., Uysal, A., & Roach, E. C. (2013). Acute phase reactants. Acta Medica, 2, 2–7.
Kim, N. D., & Luster, A. D. (2015). The role of tissue resident cells in neutrophil recruitment.
Trends in Immunology, 36, 547–555.
Kjaer, M. (2004). Role of extracellular matrix in adaptation of tendon and skeletal muscle to
mechanical loading. Physiological Reviews, 84, 649–698.
Knott, M., & Voss, D. (1968). Proprioceptive neuromuscular facilitation: Patterns and techniques.
New York: Harper & Row.
Knudson, D. (2006). The biomechanics of stretching. Journal of Exercise Science and
Physiotherapy, 2, 3–12.
Knudson, D. (2007). Fundamentals of biomechanics. New York: Springer.
Ko, K. S., & Mcculloch, C. A. (2001). Intercellular mechanotransduction: Cellular circuits that
coordinate tissue responses to mechanical loading. Biochemical and Biophysical Research
Communications, 285, 1077–1083.
Koh, T., Petersen, J. M., Pizza, F. X., & Brooks, S. V. (2003a). Passive stretches protect skeletal
muscle of adult and old mice from lengthening contraction-induced injury. The Journals of
Gerontology. Series A, Biological Sciences and Medical Sciences, 58, 592–597.
Koh, T. J., & Brooks, S. V. (2001). Lengthening contractions are not required to induce protec-
tion from contraction-induced muscle injury. American Journal of Physiology. Regulatory,
Integrative and Comparative Physiology, 281, R155–R161.
Koh, T. J., Petersen, J. M., Pizza, F. X., & Brooks, S. V. (2003b). Passive stretches protect skel-
etal muscle of adult and old mice from lengthening contraction-induced injury. Journal of
Gerontology, 58A, 592–597.
Kokkonen, J., Nelson, A. G., & Cornwell, A. (1998). Acute muscle stretching inhibits maximal
strength performance. Research Quarterly for Exercise and Sport, 69, 411–415.
Kolaczkowska, E., & Kubes, P. (2013). Neutrophil recruitment and function in health and inflam-
mation. Nature Reviews Immunology, 13, 159–175.
Kolber, M. J., Fuller, C., Marshall, J., Wright, A., & Hanney, W. J. (2012). The reliability and con-
current validity of scapular plane shoulder elevation measurements using a digital inclinometer
and goniometer. Physiotherapy Theory and Practice, 28, 161–168.
Kolber, M. J., Vega Jr., F., Widmayer, K., & Cheng, M. S. (2011). The reliability and minimal detect-
able change of shoulder mobility measurements using a digital inclinometer. Physiotherapy
Theory and Practice, 27, 176–184.
Komulainen, J., Takala, T. E. S., Kuipers, H., & Hesselink, M. K. C. (1998). The disruption of
myofibre structures in rat skeletal muscle after forced lengthening contractions. Pflügers
Archiv - European Journal of Physiology, 436, 735–741.
Konrad, A., & Tilp, M. (2014). Effects of ballistic stretching training on the properties of human
muscle and tendon structures. Journal of Applied Physiology, 117, 29–35.
Kontrogianni-Konstantopoulos, A., Ackermann, M. A., Bowman, A. L., Yap, S. V., & Bloch, R. J.
(2009). Muscle giants: Molecular scaffolds in sarcomeregenesis. Physiological Reviews, 89,
1217–1267.
Kontrogianni-Konstantopoulos, A., & Bloch, R. J. (2005). Obscurin: A multitasking muscle giant.
Journal of Muscle Research and Cell Motility, 26, 419–426.
References 111
Kontrogianni-Konstantopoulos, A., Catino, D. H., Strong, J. C., Sutter, S., Borisov, A. B., Pumplin,
D. W., et al. (2006). Obscurin modulates the assembly and organization of sarcomeres and the
sarcoplasm reticulum. The FASEB Journal, 20, 2102–2111.
Kontrogianni-Konstantopoulos, A., Jones, E. M., Van Rossum, D. B., & Bloch, R. J. (2003).
Obscurin is a ligand for small ankyrin 1 in skeletal muscle. Molecular Biology of the Cell, 14,
1138–1148.
Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T., et al. (1994).
Impaired immune and acute-phase responses in interleukin-6-deficient mice. Nature, 368,
339–342.
Kostyukova, A. S. (2008). Tropomodulins and tropomodulin/tropomyosin interactions. Cellular
and Molecular Life Sciences, 65, 563–569.
Kraushaar, B. S., & Nirschl, R. P. (1999). Current concepts review-tendinosis of the elbow (ten-
nis elbow). Clinical features and findings of histological immunohistochemical, and electron
microscopy studies. JBJS, 81, 259–278.
Krendel, M., & Mooseker, M. S. (2005). Myosins: Tails (and heads) of functional diversity.
Kresse, H., & Schonherr, E. (2001). Proteoglycans of the extracellular matrix and growth control.
Journal of Cellular Physiology, 189, 266–274.
Kruger, M., Wright, J., & Wang, K. (1991). Nebulin as a length regulator of thin filaments of ver-
tebrate skeletal muscles: Correlation of thin filament length, nebulin size, and epitope profile.
JCB, 115, 97–107.
Kuipers, H. (1994). Exercise-induced muscle damage. International Journal of Sports Medicine,
15, 132–135.
Kumamoto, T., Kleese, W. C., Cong, J., Goll, D. E., Pierce, P. R., & Allen, R. E. (1992). Localization
of the Ca2+-dependent proteinases and their inhibitor in normal, fasted, and denervated rat skel-
etal muscle. The Anatomical Record, 232, 60–77.
Kumar, C. K. K., & Chakrabarty, S. (2010). A comparative study of static stretching versus bal-
listic stretching on the flexibility of the hamstring muscles of athletes. British Journal of Sports
Medicine, 44, i16.
Kung, C. (2005). A possible unifying principle of mechanosensation. Nature, 436, 647–654.
Kunimatsu, M., Higashiyama, S., Sato, K., Ohkubo, I., & Sasaki, M. (1989). Calcium dependent
cysteine proteinase is a precursor or a chemotactic factor for neutrophils. Biochemical and
Biophysical Research Communications, 164(2), 875–882.
Kunimatsu, M., Ma, X. J., Ozaki, Y., Narita, M., Mizokami, M., & Sasaki, M. (1995). Neutrophil
chemotactic N-acetyl peptides from the calpain small subunit are also chemotactic for immu-
nocytes. Biochemistry and Molecular Biology International, 35, 247–254.
Kunimatsu, M., Ma, X. J., Ozaki, Y., Nishimura, J., Baba, S., & Sasaki, M. (1993). Neutrophil
responses induced by formyl and acetyl peptides with the N-terminal sequence of the calpain
small subunit. Biochemistry and Molecular Biology International, 31, 477–484.
Kurek, J. B., Nouri, S., Kannourakis, G., Murphy, M., & Austin, L. (1996). Leukemia inhibitory
factor and interleukin-6 are produced by diseases and regenerating skeletal muscle. Muscle &
Nerve, 19, 1291–1301.
Kushner, I. (1982). The phenomenon of the acute phase response. Annals of the New York Academy
of Sciences, 389, 39–48.
Kushner, I. (1993). Regulation of the acute phase response by cytokines. Perspectives in Biology
and Medicine, 36, 611–622.
Kushner, I., Ganapathi, M., & Schultz, D. (1989). The acute phase response is mediated by hetero-
geneous mechanisms. Annals of the New York Academy of Sciences, 557, 19–30.
Labeit, D., Watanabe, K., Witt, C., Fujita, H., Wu, Y., Lahmers, S., et al. (2003). Calcium-dependent
molecular spring elements in the giant protein titin. Proceedings of the National Academy of
Sciences of the USA, 100, 13716–13721.
Labeit, S., Gautel, M., Lakey, A., & Trinick, J. (1992). Towards a molecular understanding of titin.
The EMBO Journal, 11, 1711–1716.
Labeit, S., & Kolmerer, B. (1995). Titins: Giant proteins in charge of muscle ultrastructure and
elasticity. Science, 270, 293–296.
Labeit, S., Ottenheijm, C. A. C., & Granzier, H. (2011). Nebulin, a major player in muscle health
and disease. FASEB, 25, 822–829.
Lange, S., Ehler, E., & Gautel, M. (2006). From A to Z and back? Multicompartment proteins in
the sarcomere. Trends in Cell Biology, 16, 11–18.
Lange, S., Perera, S., Teh, P., & Chen, J. (2012). Obscurin and KCTD6 regulate cullin-dependent
small ankyrin-1 (sAnk1.5) protein turnover. Molecular Biology of the Cell, 23, 2490–2504.
Latash, M. L., & Zatsiorsky, V. M. (1993). Joint stiffness: Myth or reality? Human Movement
Science, 12, 653–692.
Lavagnino, M., Wall, M. E., Little, D., Banes, A. J., Guilak, F., & Arnoczky, S. P. (2015). Tendon
mechanobiology: Current knowledge and future research opportunities. Journal of Orthopaedic
Research, 33, 813–822.
Law, D. J. (1993). Ultrastructural comparison of slack and stretched myotendinous junctions,
based on a three-dimensional model of the connecting domain. Journal of Muscle Research
and Cell Motility, 14, 401–411.
Law, R. Y. W., Harvey, L. A., Nicholas, M. K., Tonkin, L., De Sousa, M., & Finniss, D. G. (2009).
Stretch exercises increase tolerance to stretch in patients with chronic musculoskeletal pain: A
randomized controlled trial. Physical Therapy, 89, 1016–1026.
Lawrence, T., Willoughby, D. A., & Gilroy, D. W. (2002). Anti-inflammatory lipid mediators and
insights into the resolution of inflammation. Nature Reviews Immunology, 2, 787–795.
Lazarides, E. (1980). Intermediate filaments as mechanical integrators of cellular space. Nature
(London), 283, 249–256.
Lazarides, E. (1982). Intermediate Filaments: A chemically heterogeneous developmentally regu-
lated class of proteins. Annual Review of Biochemistry, 51, 219–250.
Lea, R. D., & Gerhardt, J. J. (1995). Range-of-motion measurements. The Journal of Bone and
Joint Surgery. American Volume, 77, 784–798.
Lee, K., & Nelson, C. M. (2012). New insights into the regulation of epithelial-mesenchymal tran-
sition and tissue fibrosis. International Review of Cell and Molecular Biology, 294, 173–206.
Lefaucheur, J. P., & Sebille, A. (1995). The cellular events of injured muscle regeneration depend
on the nature of the injury. Neuromuscular Disorders, 5, 501–509.
Ley, K., Laudanna, C., Cybulsky, M. I., & Noursharhg, S. (2007). Getting to the site of inflamma-
tion: The leukocyte adhesion cascade update. Nature Reviews Immunology, 7, 678–689.
Li, Z., Mericskay, M., Agbulut, O., Butler-Browne, G., Carlson, L., Thornell, L.-E., et al. (1997).
Desmin is essential for the tensile strength and integrity of myofibrils but not for the myogenic
commitment, differentiation, and fusion of skeletal muscle. JCB, 139, 129–144.
Libby, P., Ridker, P. M., & Maseri, A. (2002). Inflammation and atherosclerosis. Circulation, 105,
1135–1143.
Lieber, R. L. (1992). Skeletal muscle structure and function: Implications for physical therapy and
sport medicine. Baltimore: Williams & Wilkins.
Lieber, R. L., & Friden, J. (1993). Muscle damage is not a function of muscle force but active
muscle strain. Journal of Applied Physiology, 74, 520–526.
Lieber, R. L., & Friden, J. (2000). Functional and clinical significance of skeletal muscle architec-
ture. Muscle & Nerve, 23, 1647–1666.
Lieber, R. L., Roberts, T. J., Blemker, S. S., Lee, S. S. M., & Herzog, W. (2017). Skeletal muscle
mechanics, energetics and plasticity. Journal of NeuroEngineering and Rehabilitation, 14,
1–16.
Liew, F. Y., & Mcinnes, I. B. (2002). Role of interleukin 15 and interleukin 18 in inflammatory
response. Annals of the Rheumatic Diseases, 61, ii100–ii102.
Light, N., & Champion, A. E. (1984). Characterization of muscle epimysium, perimysium and
endomysium. The Biochemical Journal, 219, 1017–1026.
References 113
Linke, W. A. (2008). Sense and stretchability: The role of titin and titin-associated proteins in myo-
cardial stress-sensing and mechanical dysfunction. Cardiovascular Research, 77, 637–648.
Linke, W. H., Ivemeyer, M., Mundel, P., Stockmeier, M. R., & Kolmerer, B. (1998). Nature of
PEVK-titin elasticity in skeletal muscle. Proceedings of the National Academy of Sciences of
the USA, 95, 8052–8057.
Linnemann, A., Van Der Ven, P. F., Vakeel, P., Albinus, B., Simonis, D., Bendas, G., et al. (2010).
The sarcomeric Z-disc component myopodin is a multiadapter protein that interacts with fila-
min and α-actinin. European Journal of Cell Biology, 89, 681–692.
Littlefield, R. S., & Fowler, M. D. (2008). Thin filament length regulation in striated muscle sar-
comeres: Pointed-end dynamics go beyond a nebulin ruler. Seminars in Cell & Developmental
Biology, 19, 511–519.
Liu, L., Srikakulam, R., & Winkelmann, D. A. (2008). Unc45 activates Hsp90-dependent folding
of the myosin motor domain. The Journal of Biological Chemistry, 283, 13185–13193.
Liu, X., & Spolarics, Z. (2003). Methemoglobin is a potent activator of endothelial cells by stimu-
lating IL-6 and IL-8 production and E-selectin membrane expression. American Journal of
Physiology. Cell Physiology, 285, C1036–C1046.
Llinas, P. (2015). How actin initiates the motor activity of myosin. Developmental Cell, 33,
401–412.
Lo, C. M., Wang, H. B., Dembo, M., & Wang, Y. L. (2000). Cell movement is guided by the rigidity
of the substrate. Biophysical Journal, 79, 144–152.
Lowey, S., & Trybus, K. M. (1995). Role of skeletal and smooth muscle myosin light chains.
Biophysical Journal, 68, 120s–127s.
Lu, H. H., & Jiang, J. (2006). Interface tissue engineering and the formulation of multiple tissue
systems. Advances in Biochemical Engineering/Biotechnology, 102, 91–111.
Lu, P., Takai, K., Weaver, V. M., & Werb, Z. (2011). Extracellular matrix degradation and remodel-
ing in development and disease. Cold Spring Harbor Perspectives in Biology, 3, 1–24.
Lucke, S., Hoene, A., Walschus, U., Kob, A., Pissarek, J.-W., & Schlosser, M. (2015). Acute and
chronic inflammatory reaction after implantation of different extracellular porcine dermis col-
lagen matrices in rats. BioMed Research International, 2015, 1–10.
Luft, F. C. (2006). Clinical implications. Journal of Molecular Medicine, 84, 443–445.
Lund, H., Vestergaard-Poulsen, P., Kanstrup, I.-L., & Sejrsen, P. (1998). The effect of passive
stretching on delayed onset muscle soreness, and other detrimental effects following eccentric
exercise. Scandinavian Journal of Medicine & Science in Sports, 8, 216–221.
Luther, P. K. (2009). The vertebrate muscle Z-disc: Sarcomere anchor for structure and signaling.
Journal of Muscle Research and Cell Motility, 30, 171–185.
Luttgens, K., & Hamilton, N. (1997). Kinesiology: Scientific basis of human motion. Madison, WI:
Brown & Benchmark.
Lutz, G. J., & Lieber, R. L. (1999). Skeletal muscle myosin II structure and function. Exercise and
Sport Sciences Reviews, 27, 63–77.
Macintyre, D. L., Reid, W. D., Lyster, D. M., Szasz, I. J., & Mckenzie, D. C. (1996). Presence of
WBC, decreased strength, and delayed soreness in muscle after eccentric exercise. Journal of
Applied Physiology, 80, 1006–1013.
Macintyre, D. L., Reid, W. D., & Mckenzie, D. C. (1995). The inflammatory response to muscle
injury and its clinical implications. Sports Medicine, 20, 24–40.
Mackay, B., Harrop, T. J., & Muir, A. R. (1969). The fine structure of the muscle tendon junction
in the rat. Acta Anatomica, 73, 588–604.
Mackay, D. M. (1963). Psychophysics perceived intensity: A theoretical basis for Fechner’s and
Steven’s laws. Science, 139, 1213–1216.
Maffulli, N., King, J., & Helms, P. (1994). Training in elite young athletes (the training of young
athletes (TOYA) study): Injuries, flexibility and isometric strength. British Journal of Sports
Medicine, 28, 123–136.
Magnusson, S. P. (1998). Passive properties of human skeletal muscle during stretch maneuvers-a
review. Scandinavian Journal of Medicine & Science in Sports, 8, 65–77.
Magnusson, S. P., Simonsen, E., Aagaard, P., Sorensen, H., & Kjaer, M. (1996). A mechanism for
altered flexibility in human skeletal muscle. Journal of Physiology (London), 497, 291–298.
Magnusson, S. P., Simonsen, E., Dyhre-Poulsen, P., Aagaard, P., Mohr, T., & Kjaer, M. (1996).
Viscoelastic stress relaxation during static stretch in human skeletal muscle in the absence of
EMG activity. Medicine & Science in Sports & Exercise, 6, 323–328.
Mahieu, N. N., Mcnair, P., De Muynck, M., Stevens, V., Blanckaert, I., Smits, N., et al. (2007).
Effect of static and ballistic stretching on the muscle-tendon tissue properties. Medicine and
Science in Sports and Exercise, 39, 494–501.
Makino, A., Shin, H. Y., Komai, Y., Fukuda, S., Coughlin, M., Sugihara-Seki, M., et al. (2007).
Mechanotransduction in leukocyte activation: A review. Biorheology, 44, 221–249.
Malliaropoulos, N., Papalexandris, S., Papalada, A., & Papacostas, E. (2004). The role of stretch-
ing in rehabilitation of hamstring injuries: 80 athletes follow-up. Medicine and Science in
Malm, C., Nyberg, P., Engstrom, M., Sjodin, B., Lenkei, R., & Ekblom, B. (2000). Immunological
changes in human skeletal muscle and blood after eccentric exercise and multiple biopsies. The
Mammoto, T., & Ingber, D. E. (2010). Mechanical control of tissue and organ development.
Development, 137, 1407–1420.
Maniotis, A. J., Chen, C. S., & Ingber, D. E. (1997). Demonstration of mechanical connections
between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure.
Margadant, F., Chew, L. L., Hu, X., Yu, H., Bate, N., Zhang, X., et al. (2011). Mechanotransduction
in vivo by repeated talin stretch-relaxation events depends upon vinculin. PLoS Biology, 9,
e1001223.
Marino, A., & Giotta, N. (2008). Cinacalcet, fetuin-A and interleukin-6. Nephrology, Dialysis,
Transplantation, 23, 1461.
Marino, M., Scuderi, F., Provenzano, C., & Bartoccioni, E. (2011). Skeletal muscle cells: From
local inflammatory response to active immunity. Gene Therapy, 18, 109–116.
Marmelzat, W. L. (1977). Medicine and history-the contributions to dermatologic surgery of
Aulus Cornelius Celsus (Circa 30 B.C. - A.D. 50). The Journal of Dermatologic Surgery and
Oncology, 3, 161–166.
Marsolais, D., Cote, C. H., & Frenette, J. (2001). Neutrophils and macrophages accumulate sequen-
tially following Achilles tendon injury. Journal of Orthopaedic Research, 19, 1203–1209.
Martinez, F. O., Helming, L., & Gordon, S. (2009). Alternative activation of macrophages: An
immunologic functional perspective. Annual Review of Immunology, 27, 451–483.
Martinez, F. O., Sica, A., Mantovani, A., & Locati, M. (2008). Macrophage activation and polariza-
tion. Frontiers in Bioscience, 13, 453–461.
Martins, W. R., Carvalho, M. M., Mota, M. R., Cipriano, G. F. B., Mendes, F. A. S., Diniz, L. R.,
et al. (2013). Diacutaneous fibrolysis versus passive stretching after articular immobilisation:
Muscle recovery and extracellular matrix remodelling. Osteoarthritis Medical Hypotheses, 1,
17–22.
Maruyama, K., Kimura, S., Ohashi, K., & Kuwano, Y. (1981). Connectin, an elastic protein of
muscle, identification of “titin” with connectin. Journal of Biochemistry, 89, 701–709.
Massimino, M. L., Rapizzi, E., Cantini, M., Libera, L. D., Mazzoleni, F., Arslan, P., et al. (1997).
ED2+ macrophages increase selectively myoblast proliferation in muscle cultures. Biochemical
and Biophysical Research Communications, 235, 75759.
Mastorakos, G., Chrousos, G. P., & Weber, J. S. (1993). Recombinant interleukin-6 activates the
hypothalamic-pituitary-adrenal axis in humans. The Journal of Clinical Endocrinology and
Metabolism, 77, 1690–1694.
Maturana, H. R., & Varela, F. J. (1975). Autopoietic systems. Champaign, IL: University of Illinois
BLC Report 9.
Mayer, U. (2003). Integrins: Redundant or important players in skeletal muscle. JBC, 278,
14587–14590.
References 115
Mayerson, N. H., & Milano, R. A. (1984). Goniometric measurement reliability in physical medi-
cine. Archives of Physical Medicine and Rehabilitation, 65, 92–94.
Mazzochi, F. (2008). Complexity in biology-Exceeding the limits of reductionism and determin-
ism using complexity theory. EMBO Reports, 9, 10–14.
Mcburney, D. H., & Balaban, C. D. (2009). A heuristic model of sensory adaptation. Attention,
Perception, & Psychophysics, 71, 1941–1961.
Mccall, G. E., Byrnes, W. C., Dickinson, A., Pattany, P. M., & Fleck, S. J. (1996). Muscle fiber
hypertrophy, hyperplasia, and capillary density in college men after resistance training. Journal
of Applied Physiology, 81, 2004–2012.
Mcclure, P., Blackburn, L., & Dusold, C. (1994). The use of splints in the treatment of joint stiff-
ness: Biologic rationale and an algorithm for making clinical decisions. Physical Therapy, 74,
1101–1107.
Mcelhinny, A. S., Kazmierski, S. T., Labeit, S., & Gregorio, C. C. (2003). Nebulin: The nebulous
multifunctional giant of striated muscle. Trends in Cardiovascular Medicine, 13, 195–201.
Mcelhinny, A. S., Kolmerer, B., Fowler, V. M., Labeit, S., & Gregorio, C. C. (2001). The
N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments.
The Journal of Biological Chemistry, 276, 583–592.
Mcewen, B. S., & Wingfield, J. C. (2003). The concept of allostasis in biology and biomedicine.
Hormones and Behavior, 43, 2–15.
Mcgee, D. W., Bamberg, T., Vitukus, S. J. D., & Mcghee, J. R. (1995). A synergistic relationship
between TNF-α, IL-1β, and TGF-β1 on IL-6 secretion by the IEC-6 intestinal epithelial cell
line. Immunology, 86, 6–11.
Mclachlan, A. D., & Karn, J. (1982). Periodic charge distributions in the myosin rod amino acid
sequence match cross-bridge spacings in muscle. Nature, 299, 226–231.
Mclennan, I. S. (1993). Resident macrophages (ED2- and ED3-positive) do not phagocytose
degenerating rat skeletal muscle fibres. Cell and Tissue Research, 272, 193–196.
Mclennan, I. S. (1996). Degenerating and regenerating skeletal muscles contain several subpopu-
lations of macrophages with distinct spatial and temporal distributions. Journal of Anatomy,
188, 17–28.
Mcloughlin, T. J., Mylona, E., Hornberger, T. A., Esser, K. A., & Pizza, F. X. (2003). Inflammatory
cells in rat skeletal muscle are elevated after electrically stimulated contractions. Journal of
Mcmahon, T. A. (1984). Muscles, reflexes, and locomotion. Princeton, NJ: Princeton University
Press.
Mcnulty, R. J. (2006). Fibroblasts and myofibroblasts: Their source, function and role in disease.
The International Journal of Biochemistry & Cell Biology, 39, 666–671.
Medhitov, R. (2008). Origin and physiological roles of inflammation. Nature, 454, 428–435.
Medzhitov, R. (2010). Inflammation 2010: New adventures of an old flame. Cell, 140, 771–771.
Meier-Ewert, H. K., Ridker, P. M., Rifai, N., Price, N., Dinges, D. F., & Mullington, J. M. (2001).
Absence of diurnal variation of C-reactive protein concentrations in healthy human subjects.
Clinical Chemistry, 47, 426–430.
Meredith Jr., J. E., Fazeli, B., & Schwartz, M. A. (1993). The extracellular matrix as a cell survival
factor. Molecular Biology of the Cell, 4, 953–961.
Messner, K., Wei, Y., Andersson, B., Gillquist, J., & Rasanen, T. (1999). Rat model of Achilles
tendon disorder. A pilot study. Cells, Tissues, Organs, 165, 30–39.
Metsios, G. S., Stavropoulos-Kalinoglou, A., & Kitas, G. D. (2015). The role of exercise in the
management of rheumatoid arthritis. Expert Review of Clinical Immunology, 11, 1121–1130.
Meyer, L. C., & Wright, N. T. (2013). Structure of giant muscle proteins. Frontiers in Physiology,
4, 1–12.
Michele, D. E., & Campbell, K. P. (2003). Dystrophin-glycoprotein complex: Post-translational
processing and dystroglycan function. JBC, 278, 15457–15460.
Miller, B. F., Olesen, J. L., Hansen, M., Dossing, S., Crameri, R. M., Welling, R. J., et al. (2005).
Coordinated collagen and muscle protein synthesis in human patella tendon and quadriceps
muscle after exercise. The Journal of Physiology, 567, 1021–1033.
Miller, M. K., Bang, M.-L., Witt, C. C., Labeit, D., Trombitas, C., Watanabe, K., et al. (2003). The
muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-
based stress response molecules. Journal of Molecular Biology, 333, 951–964.
Milz, S., Ockert, B., & Putz, R. (2009). Tenocytes and the extracellular matrix: A reciprocal rela-
tionship. Orthopade, 38, 1071–1079.
Minutti, C. M., Knipper, J. A., Allen, J. E., & Zaiss, D. M. W. (2017). Tissue-specific contribution
of macrophages to wound healing. Seminars in Cell & Developmental Biology, 61, 3–11.
Mitroulis, I., Alexaki, V. I., Kourtzelis, I., Ziogas, A., Hajishengallis, G., & Chavakis, T. (2015).
Leukocyte integrins: Role in leukocyte recruitment and as therapeutic targets in inflammatory
disease. Pharmacology & Therapeutics, 147, 123–135.
Mittal, M., Siddiqui, R., Tran, K., Reddy, S. P., & Malik, A. B. (2014). Reactive oxygen species in
inflammation and tissue injury. Antioxidants & Redox Signaling, 20, 1126–1166.
Moore, M. A., & Hutton, R. S. (1980). Electromyographic investigation of muscle stretching tech-
niques. Medicine and Science in Sports and Exercise, 12, 322–329.
Morcelli, M. H., Oliveira, J. M. C. A., & Navega, M. T. (2013). Comparison of static, ballistic and
contract-relax stretching in hamstring muscle. Fisioterapia e Pesquisa, 20, 244–249.
Moreau, N. G., Simpson, K. N., Teefey, S. A., & Diamano, D. L. (2010). Muscle architecture
predicts maximum strength and is related to activity levels in cerebral palsy. Physical Therapy,
90, 1619–1630.
Morozov, V. I., Tsyplenkov, P. V., Goldberg, N. D., & Kalinski, M. I. (2006). The effects of high-
intensity exercise on skeletal muscle neutrophil myeloperoxidase in untrained and trained rats.
European Journal of Applied Physiology, 97, 716–722.
Mueller, M. J., & Maluf, K. (2002). Tissue adaptation to physical stress: A proposed “physical
stress theory” to guide physical therapist practice, education, and research. Physical Therapy,
82, 383–403.
Mujika, I., Chatard, J.-C., Busso, T., Geyssant, A., Barale, F., & Lacoste, L. (1995). The effects of
training on performance in competitive swimming. Canadian Journal of Applied Physiology,
20, 395–406.
Muller, W. A. (2013). Getting leukocytes to the site of inflammation. Veterinary Pathology, 50,
7–22.
Murphy, D. R. (1994). A critical look at static stretching: Are we doing our patients. Chiropractic
Murphy, R. M. (2009). Calpains, skeletal muscle function and exercise. Proceedings of the
Australian Physiological Society, 40, 95–102.
Murray, P. J., & Wynn, T. A. (2011). Protective and pathogenic functions of macrophage subsets.
Nature Reviews Immunology, 11, 723–737.
Nakamura, M., Ikezoe, T., Takeno, Y., & Ichihashi, N. (2012). Effects of a 4-week static stretch
training program on passive stiffness of human gastrocnemius muscle-tendon unit in vivo.
Nathan, C., & Ding, A. (2010). Nonresolving inflammation. Cell, 140, 871–882.
Nelson, C. M., & Bissell, M. J. (2006). Of extracellular matrix, scaffolds, and signaling: Tissue
architecture regulates development, homeostasis, and cancer. Annual Review of Cell and
Developmental Biology, 22, 287–309.
Newham, D. J., Jones, D. A., & Edwards, R. H. T. (1983). Large delayed plasma creatine kinase
changes after stepping exercise. Muscle & Nerve, 6, 380–385.
Newham, D. J., Mcphail, G., Mills, K. R., & Edwards, R. H. T. (1983). Ultrastructural change after
concentric and eccentric contractions of human muscle. Journal of Neurosurgical Sciences,
61, 109–122.
Ng, I. C. L., Maull, R., & Yip, N. (2009). Outcome-based contracts as a driver for systems thinking
and service-dominant logic in service science: Evidence from the defence industry. European
Management Journal, 27, 377–387.
Nicola, N. A. (1994). Cytokine pleiotropy and redundancy: A view from the receptor. Stem Cells,
12, 3–12.
References 117
Nielsen, A. R., Mounier, R., Plomgaard, P., Mortensen, O. H., Penkowa, M., Speerschneider, T.,
et al. (2007). Expression of interleukin-15 in human skeletal muscle - effect of exercise and
muscle fibre type composition. The Journal of Physiology, 584, 305–312.
Nieman, D. C. (1997). Immune response to heavy exercise. Journal of Applied Physiology, 82,
1385–1394.
Nieman, D. C., Nehlsen-Cannarella, S. L., Fagoaga, O. R., Henson, D. A., Utter, A., Davis, M. J.,
et al. (1998). Influence of mode and carbohydrate on the cytokine response to heavy exertion.
Medicine and Science in Sports and Exercise, 30, 671–678.
Nikolaou, P. K., Macdonald, B. L., Glisson, R. R., Seaber, A. V., & Garrett Jr., W. E. (1987).
Biomechanical and histological evaluation of muscle after controlled strain injury. The
American Journal of Sports Medicine, 15, 9–14.
Nishikawa, K. C., Monroy, J. A., Uyeno, T. E., Yeo, S. H., Pai, D. K., & Lindstedt, S. L. (2012). Is
titin a ‘winding filament’? A new twist on muscle contraction. Proceedings of the Royal Society
B: Biological Sciences, 279, 981–990.
Novak, M. L., & Koh, T. J. (2013). Phenotype transitions of macrophages orchestrate tissue repair.
The American Journal of Pathology, 183, 1352–1363.
Nussbaumer, S., Leunig, M., Glatthorn, J. F., Stauffacher, S., Gerber, H., & Maffiuletti, N. A.
(2010). Validity and test-retest reliability of manual goniometers for measuring passive hip
range of motion in femoroacetabular impingement patients. BMC Musculoskeletal Disorders,
11, 1–11.
O’hara, J., Cartwright, A., Wade, C. D., Hough, A. D., & Shum, G. L. (2011). Efficacy of static
stretching and proprioceptive neuromuscular facilitation stretch on hamstrings length after a
single session. Journal of Strength and Conditioning Research, 25, 1586–1591.
Offer, G., & Sessions, R. (1995). Computer modelling of the a-helical coiled coil: Packing of side-
chains in the inner core. Journal of Molecular Biology, 249, 967–987.
Ohlendieck, K. (1996). Towards an understanding of the dystrophin-glycoprotein complex:
Linkage between the extracellular matrix and the membrane cytoskeleton in muscle fibers.
European Journal of Cell Biology, 69, 1–10.
Oiwa, K., & Manstein, D. J. (Eds.). (2007). Controlled nanoscale motion. Berlin: Springer.
Oltvai, Z. N., & Barabasi, A.-L. (2002). Life’s complexity pyramid. Science, 298, 763–764.
Orimo, S., Hiyamuta, E., Arahata, K., & Sugita, H. (1991). Analysis of inflammatory cells and
complement C3 in bupivacaine-induced myonecrosis. Muscle & Nerve, 14, 515–520.
Orr, A. W., Helmke, B. P., Blackman, B. R., & Schwartz, M. A. (2006). Mechanisms of mechano-
transduction. Developmental Cell, 10, 11–20.
Ostapiuk-Karolczuk, J., Zembron-Lacny, A., Naczk, M., Gajewski, M., Kasperska, A., Dziewiecka,
H., et al. (2012). Cytokines and cellular inflammatory sequence in non-athletes after prolonged
exercise. The Journal of Sports Medicine and Physical Fitness, 52, 563–568.
Osternig, L. R., Robertson, R., Troxel, R., & Hansen, P. (1987). Muscle activation during proprio-
ceptive neuromuscular facilitation (PNF) stretching techniques. American Journal of Physical
Medicine & Rehabilitation, 66, 298–307.
Ostrowski, K., Rohde, T., Asp, S., Schjerling, P., & Pedersen, B. K. (1999). Pro- and anti-
inflammatory cytokine balance in strenuous exercise in humans. The Journal of Physiology,
515, 287–291.
Ostrowski, K., Rohde, T., Zacho, M., Asp, S., & Pedersen, B. K. (1998). Evidence that interleukin-
6 is produced in human skeletal muscle during prolonged running. The Journal of Physiology,
508, 949–953.
Ostrowski, K., Schjerling, P., & Pedersen, B. K. (2000). Physical activity and plasma interleukin-
6 in humans - effect of intensity of exercise. European Journal of Applied Physiology, 83,
512–515.
Ottenheijm, C. A., & Granzier, H. (2010). Lifting the nebula: Novel insights into skeletal muscle
contractility. Physiology (Bethesda), 25, 304–310.
Page, P. (2012). Current concepts in muscle stretching for exercise and rehabilitation. IJSPT, 7,
109–119.
Palmer, J. F. (1835). The works of John Hunter, F.R.S. with notes. London: Longman, Rees, Orme,
Brown, Green, & Longman.
Palmisano, M. G., Bremner, S. N., Hornberger, T. A., Meyer, G. A., Domenighetti, A. A., Shah,
S. B., et al. (2015). Skeletal muscle intermediate filaments form a stress-transmitting and
stress-signaling network. Journal of Cell Science, 128, 219–224.
Paluch, E. K., Nelson, C. M., Biais, N., Fabry, B., Moeller, J., Pruitt, B. L., et al. (2015).
Mechanotransduction: Use the force(s). BMC Biology, 13, 1–14.
Papadimitriou, J. M., Robertson, T. A., Mitchell, C. A., & Grounds, M. D. (1990). The process of
new plasmalemma formation in focally injured skeletal muscle fibers. Journal of Structural
Biology, 103, 124–134.
Pardo, J. V., D’Angelo Siliciano, J., & Craig, S. W. (1983). A vinculin-containing cortical lattice in
skeletal muscle: Transverse lattice elements (“costameres”) mark sites of attachment between
myofibrils and sarcolemma. Proceedings of the National Academy of Sciences of the USA, 80,
1008–1012.
Peake, J. M., Nosaka, K., & Suzuki, K. (2005). Characterization of inflammatory responses to
eccentric exercise in humans. Exercise Immunology Review, 11, 64–85.
Peake, J. M., Suzuki, K., Hordern, M., Wilson, G., Nosaka, K., & Coombes, J. S. (2005). Plasma
cytokine changes in relation to exercise intensity and muscle damage. European Journal of
Pedersen, B. K. (2006). The anti-inflammatory effect of exercise: Its role in diabetes and cardio-
vascular disease control. Essays in Biochemistry, 42, 105–117.
Pedersen, B. K., Akerstrom, T. C. A., Nielsen, A. R., & Fischer, C. P. (2007). Role of myokines in
exercise and metabolism. Journal of Applied Physiology, 103, 1093–1098.
Pedersen, B. K., & Febbraio, M. A. (2008). Muscle as an endocrine organ: Focus on muscle
derived interleukin-6. Physiological Reviews, 88, 1379–1406.
Pedersen, B. K., & Fischer, C. P. (2007). Beneficial health effects of exercise - The role of IL-6 as
a myokine. Trends in Pharmacological Sciences, 28, 152–156.
Pedersen, B. K., & Hoffman-Goetz, L. (2000). Exercise and the immune system: Regulation, inte-
gration and adaption. Physiological Reviews, 80, 1055–1081.
Pedersen, B. K., Ostrowski, K., Rohde, T., & Bruunsgaard, H. (1998). The cytokine response to
strenuous exercise. Canadian Journal of Physiology and Pharmacology, 76, 505–511.
Pedersen, B. K., Steensberg, A., Fischer, C. P., Keller, C., Ostrowski, K., & Schjerling, P. (2001).
Exercise and cytokines with particular focus on muscle-derived IL-6. Exercise Immunology
Review, 7, 18–31.
Pepys, M. B., & Hirschfield, G. M. (2003). C-reactive protein: A critical update. The Journal of
Clinical Investigation, 111, 1805–1812.
Perrimon, N., & Bernfield, M. (2001). Cellular functions of proteoglycans-an overview. Seminars
in Cell & Developmental Biology, 12, 65–67.
Peter, A. K., Cheng, H., Ross, R. S., Knowlton, K. U., & Chen, J. (2011). The costamere bridges
sarcomeres to the sarcolemma in striated muscle. Progress in Pediatric Cardiology, 31, 83–88.
Pfuhl, M., Winder, S. J., & Pastore, A. (1994). Nebulin, a helical actin binding protein. The EMBO
Journal, 13, 1782–1789.
Philippou, A., Maridaki, M., Theos, A., & Koutsilieris, M. (2012). Cytokines in muscle damage.
Advances in Clinical Chemistry, 58, 49–87.
Phillipson, M., Heit, B., Colarusso, P., Liu, L., Ballantyne, C. M., & Kubes, P. (2006). Intramural
crawling of neutrophils to emigration sites: A molecularly distinct process from adhesion in the
recruitment cascade. JEM, 203, 2569–2575.
Pickup, M. W., Mouw, J. K., & Weaver, V. M. (2014). The extracellular matrix modulates the hall-
marks of cancer. EMBO, 15, 1243–1253.
Physiology, 92, 1873–1878.
Pizza, F. X., Petersen, J. M., Baas, J. H., & Koh, T. J. (2005). Neutrophils contribute to mus-
cle injury and impair its resolution after lengthening contractions in mice. The Journal of
References 119
Plow, E. F., Haas, T. A., Zhang, L., Loftus, J., & Smith, J. W. (2000). Ligand binding to integrins.
JBC, 275, 21785–21788.
Porat, R., Poutsiaka, D. D., Miller, L. C., Granowitz, E. V., & Dinarello, C. A. (1992). Interleukin-1
(IL-1) receptor blockade reduces endotoxin and Borrelia burgdorferi-stimulated IL-8 synthesis
in human mononuclear cells. FASEB, 6, 2482–2486.
Powell, P. L., Roy, R. R., Kanim, P. M., Bello, A., & Edgerton, V. R. (1984). Predictability of
skeletal muscle tension from architectural determinations in guinea pig hindlimbs. Journal of
Power, K., Behm, D., Farrel, C., Carroll, M., & Young, W. (2004). An acute bout of static stretch-
ing: Effects on force and jumping performance. Medicine and Science in Sports and Exercise,
36, 1389–1396.
Powers, K., Nishikawa, K., Joumaa, V., & Herzog, W. (2016). Decreased force enhancement in
skeletal muscle sarcomeres with a deletion in titin. The Journal of Experimental Biology, 219,
1311–1316.
Prentice, S. F. (1985). Warming-up and stretching for improved physical performance and
prevention of sports related injuries. Sports Medicine, 2, 267–278.
Previtera, M. L. (2004). Mechanotransduction in the immune system. Cellular and Molecular
Bioengineering, 7, 473–481.
Proske, U., Morgan, D. L., & Gregory, J. (1993). Thixotropy in skeletal muscle and in muscle
spindles: A review. Progress in Neurobiology, 41, 704–721.
Puklin-Faucher, E., & Sheetz, M. P. (2009). The mechanical integrin cycle. JCS, 122, 179–186.
Puri, K. D., Doggett, T. A., Huang, C.-Y., Douangpanya, J., Hayflick, J. S., Turner, M., et al.
(2005). The role of endothelial PI3Kγ activity in neutrophil trafficking. Blood, 106, 150–157.
Purslow, P. (Ed.). (2008). The extracellular matrix of skeletal and cardiac muscle. Boston, MA:
Springer.
Qi, J., Fox, A. M., Alexopoulos, L. G., Chi, L., Bynum, D., Guilak, F., et al. (2006). IL-1β decreases
the elastic modulus of human tenocytes. Journal of Applied Physiology, 101, 189–195.
Quinones, S. R., Neblock, D. S., & Berg, R. A. (1986). Regulation of collagen production and
collagen mRNA amounts in fibroblasts in response to culture conditions. Biochemical Journal,
239, 179–183.
Raj, D. A., Booker, T. S., & Belcastro, A. N. (1998). Striated muscle calcium-stimulated cyste-
ine protease (calpain-like) activity promotes myeloperoxidase activity with exercise. Pflügers
Archiv - European Journal of Physiology, 435, 804–809.
Ralphs, J. R., Waggett, A. D., & Benjamin, M. (2002). Actin stress fibres and cell-cell adhesion
molecules in tendons: Organisation in vivo and response to mechanical loading of tendon cells
in vitro. Matrix Biology, 21, 67–74.
Ramadori, G., Van Damme, J., Rieder, H., & Meyer Zum Buschenfelde, K. H. (1988). Interleukin
6, the third mediator of acute-phase reaction, modulates hepatic protein synthesis in human and
mouse. Comparison with interleukin 1 beta and tumor necrosis factor-alpha. European Journal
of Immunology, 18, 1259–1264.
Randazzo, D., Giacomello, E., Lorenzini, S., Rossi, D., Pierantozzi, E., Blaauw, B., et al. (2013).
Obscurin is required for ankyin B-dependent dystrophin localization and sarcolemma integrity.
JCB, 200, 523–536.
Ransone, J., Geissler, G., Wilson, P., & Adams, B. (2012). Chapter 9: Soft tissue damage and
healing. Monaco: IAAF.
Rather, L. J. (1971). Disturbance of function (Functio Laesa): The legendary fifth cardinal sign
of inflammation, added by Galen to the four cardinal signs of Celsus. Bulletin of the New York
Academy of Medicine, 47, 303–322.
Rayment, I., Rypniewski, W. R., Schmidt-Base, K., Smith, R., Tomchick, D. R., Benning, M. M.,
et al. (1993). Three-dimensional structure of myosin subfragment-1: A molecular motor.
Science, 261, 50–58.
Reihmane, D., & Dela, F. (2013). Interleukin-6: Possible biological roles during exercise. European
Journal of Sport Science, 14, 242–250.
Ricard-Blum, S. (2011). The collagen family. Cold Spring Harbor Perspectives in Biology, 2011,
1–19.
Ricard-Blum, S., & Ruggiero, F. (2005). The collagen superfamily: From teh extracellular matrix
to the cell membrane. Pathologie Biologie, 53, 430–442.
Ritchie, D. G. (1990). Interleukin 6 stimulates hepatic glucose release from prelabeled glycogen
pools. American Journal of Physiology. Endocrinology and Metabolism, 258, E57–E64.
Robertson, T. A., Maley, M. A. L., Grounds, M. D., & Papadimitriou, J. M. (1993). The role
of macrophages in skeletal muscle regeneration with particular reference to chemotaxis.
Experimental Cell Research, 207, 321–331.
Rock, K. L., & Kono, H. (2008). The inflammatory response to cell death. Annual Review of
Pathology, 3, 99–126.
Rodgers, U. R., & Weiss, A. S. (2005). Cellular interactions with elastin. Pathologie Biologie, 53,
390–398.
Ross, T. D., Coon, B. G., Yun, S., Baeyens, N., Tanaka, K., Ouyang, M., et al. (2013). Integrins in
mechanotransduction. Current Opinion in Cell Biology, 25, 613–618.
Rothstein, J. M., Miller, P. J., & Roettger, R. F. (1983). Goniometric reliability in a clinical setting:
Elbow and knee measurement. Physical Therapy, 63, 1611–1615.
Roy, R. R., & Edgerton, V. R. (Eds.). (1992). Skeletal muscle architecture and performance.
Oxford, UK: Blackwell Scientific Publications.
Rubtsov, A. M., & Lopina, O. D. (2000). Ankyrins. FEBS Letters, 482, 1–5.
Rui, Y., Bai, J., & Perrimon, N. (2010). Sarcomere formation occurs by the assembly of multiple
latent protein complexes. PLoS Genetics, 6, 1–13.
Ruoslahti, E., & Pierschbacher, M. D. (1988). New perspectives in cell adhesion: RGD and integ-
rins. Science, 238, 491–497.
Sady, S. P., Wortman, M., & Blanke, D. (1982). Flexibility training: Ballistic, static or proprio-
ceptive neuromuscular facilitation? Archives of Physical Medicine and Rehabilitation, 63,
261–263.
Safran, M., Seaber, A., & Garrett, W. (1989). Warm-up and muscular injury prevention: An update.
Saido, T. C., Sorimachi, H., & Suzuki, K. (1994). Calpain: New perspectives in molecular diversity
and physiological-pathological involvement. FASEB, 8, 814–822.
Salvini, T. F., Durigan, J. L. Q., Peviani, S. M., & Russo, T. L. (2012). Effects of electrical stimula-
tion and stretching on the adaptation of denervated skeletal muscle - implications for physical
therapy. Revista Brasileira de Fisioterapia, 16, 175–183.
Samols, D., Agrawal, A., & Kushner, I. (2002). Acute phase proteins. Cytokine Reference, 1–16.
Sanger, J. M., & Sanger, J. W. (2008). The dynamic Z bands of striated muscle cells. Science
Signaling, 12, 1–4.
Sanger, J. W., Kang, S., Siebrands, C. C., Freeman, N., Du, A., Wang, J., et al. (2005). How to build
a myofibril. Journal of Muscle Research and Cell Motility, 26, 343–354.
Sanger, J. W., & Sanger, J. M. (2001). Fishing out proteins that bind titin. JCB, 154, 21–24.
Sanger, J. W., Sanger, J. M., & Franzini-Armstrong, C. (Eds.). (2004). Assembly of the skeletal
muscle cell. New York: McGraw-Hill.
Sapir, L., & Tzlil, S. (2017). Talking over the extracellular matrix: How do cells communicate
mechanically? Seminars in Cell & Developmental Biology, 71, 99–105.
Scapini, P., Lapinet-Vera, J. A., Gasperini, S., Calzettie, F., Bazzoni, F., & Casatella, M. A. (2000).
The neutrophil as a cellular source of chemokines. Immunological Reviews, 177, 195–203.
Schenkel, A. R., Mamdouh, Z., & Muller, W. A. (2004). Locomotion of monocytes on endothelium
is a critical step during extravasation. Nature Immunology, 5, 393–400.
Schiaffino, S., & Hanzlikova, V. (1970). On the mechanisms of compensatory hypertrophy in skel-
etal muscle. Experientia, 26, 152–153.
Schulkin, J. (2004). Allostasis, homeostasis, and the costs of physiological. Cambridge, UK:
Cambridge University Press.
Schutt, C. E., & Lindberg, U. (1992). Actin as the generator of tension during muscle contraction.
References 121
Schwartz, M. A. (2010). Integrins and extracellular matrix in mechanotransduction. Cold Spring
Harbor Perspectives in Biology, 2010, 1–13.
Schweitzer, S. C., Klymkowsky, M. W., Bellin, R. M., Robson, R. M., Capetanaki, Y., & Evans,
R. M. (2001). Paranemin and the organization of desmin filament networks. JCS, 114,
1079–1089.
Scott, A., Khan, K. M., Cook, J. L., & Duronio, V. (2004). What do we mean by the term “inflam-
mation”? A contemporary science update for sports medicine. British Journal of Sports
Medicine, 38, 372–380.
Sebestyen, M. G., Fritz, J. D., Wolff, J. A., & Greaser, M. L. (1996). Primary structure of the kinase
domain region of rabbit skeletal and cardiac muscle titin. Journal of Muscle Research and Cell
Motility, 17, 343–348.
Seiler, S. (2010). What is best practice for training intensity and duration distribution in endurance
athletes? International Journal of Sports Physiology and Performance, 5, 276–291.
Selkirk, S. B. (2000). Proteoglycans and pattern formation-sugar biochemistry meets developmen-
tal genetics. Trends in Genetics, 16, 206–212.
Shah, S. B., Davis, J., Weisleder, N., Kostavassili, I., Mcculloch, A. D., Ralston, E., et al. (2004).
Structural and functional roles of Desmin in mouse skeletal muscle during passive deforma-
tion. Biophysical Journal, 86, 2993–3008.
Shah, S. B., Su, F.-C., Jordan, K., Milner, D. J., Friden, J., Capetanaki, Y., et al. (2002). Evidence
for increased myofibrillar mobility in desmin-null mouse skeletal muscle. The Journal of
Experimental Biology, 205, 321–325.
Sharma, P., & Maffulli, N. (2006). Biology of tendon injury: Healing, modeling and remodeling.
The Journal of Musculoskeletal and Neuronal Interactions, 6, 181–190.
Sharman, M. J., Cresswell, A. G., & Riek, S. (2006). Proprioceptive neuromuscular facilitation
stretching - mechanisms and clinical implications. Sports Medicine, 36, 929–939.
Shek, P. N., & Shephard, R. J. (1998). Physical exercise as a human model of limited inflammatory
response. Canadian Journal of Physiology and Pharmacology, 76, 589–597.
Shellock, F. G., & Prentice, W. E. (1985). Warming-up and stretching for improved physical per-
formance and prevention of sports related injuries. Sports Medicine, 2, 267–278.
Shimizu, Y., & Shaw, S. (1991). Lymphocyte interactions with extracellular matrix. The FASEB
Journal, 5, 2292–2299.
Shrier, I., & Gossal, K. (2000). Myths and truths of stretching. The Physician and Sportsmedicine,
28, 1–10.
Silver, F. H. (2006). Mechanosensing and mechanochemical transduction in extracellular matrix.
New York: Springer.
Sindrilaru, A., Peters, T., Wieschalka, S., Baican, C., Baican, A., Peter, H., et al. (2011). An unre-
strained proinflammatory M1 macrophage population induced by iron impairs wound healing
in humans and mice. The Journal of Clinical Investigation, 121, 985–997.
Sjoblom, B., Salmazo, A., & Djinovic-Carugo, K. (2008). α-Actinin structure and regulation.
Cellular and Molecular Life Sciences, 65, 2688–2701.
Slauson, D. O., & Cooper, B. J. (2002). Mechanisms of disease: A textbook of comparative pathol-
ogy. St. Louis, MO: Mosby.
Small, K., Mcnaughton, L., & Matthews, M. (2008). A systematic review into the efficacy of static
stretching as part of a warm-up for the prevention of exercise-related injury. Research in Sports
Medicine, 16, 213–231.
Smith, C., Kruger, M. J., Smith, R. M., & Myburgh, K. H. (2008). The inflammatory response to
skeletal muscle injury: Illuminating complexities. Sports Medicine, 38, 947–969.
Smith, C. A. (1994). The warm-up procedure: To stretch or not to stretch. A brief review. The
Journal of Orthopaedic and Sports Physical Therapy, 19, 12–17.
soreness? Medicine and Science in Sports and Exercise, 23, 542-551.
Smith, L. L., Anwar, A., Fragen, M., Rananto, C., Johnson, R., & Holbert, D. (2000). Cytokines
and cell adhesion molecules associated with high-intensity eccentric exercise. European
Journal of Applied Physiology, 82, 61–67.
Smith, L. L., Brunetz, M., Chenier, T., Mccammon, M., Houmard, J., Franklin, M., et al. (1993).
The effects of static and ballistic stretching on delayed onset muscle soreness and creatine
kinase. Research Quarterly for Exercise and Sport, 64, 103–107.
Smith, L. R., Meyer, G. A., & Lieber, R. L. (2013). Systems analysis of biological networks in
skeletal muscle function. Wiley Interdisciplinary Reviews. Systems Biology and Medicine, 5,
55–71.
Song, W. K., Wang, W. C., Foster, R. F., Biesler, D. A., & Kaufman, S. J. (1992). H36-alpha 7 is a
novel integrin alpha chain that is developmentally regulated during skeletal myogenesis. JCB,
117, 643–657.
Sorichter, S., Puschendorf, B., & Mair, J. (1999). Skeletal muscle injury by eccentric muscle action:
Muscle protein as markers of muscle fiber injury. Exercise Immunology Review, 5, 5–21.
Sorimachi, H., Freiburg, A., Kolmerer, B., Ishiura, S., Stier, G., Gregorio, C. C., et al. (1997).
Tissue-specific expression and α-actinin binding properties of the Z-disc titin: Implications for
the nature of vertebrate Z-discs. Journal of Molecular Biology, 270, 688–695.
Soslowsky, L. J., Thomopoulos, S., Tun, S., Flanagan, C. L., Keefer, C. C., Mastaw, J., et al.
(2000). Overuse activity injures the supraspinatus tendon in an animal model: A histologic and
biomechanical study. Journal of Shoulder and Elbow Surgery, 9, 79–84.
Sothern, R. B., Roitman-Johnson, B., Kanabrocki, E. L., Yager, J. G., Roodell, M. M., Weatherbee,
J. A., et al. (1995). Circadian characteristics of circulating interleukin-6 in men. The Journal of
Allergy and Clinical Immunology, 95, 1029–1035.
Spencer, M. J., Lu, B., & Tidball, J. G. (1996). Calpain II expression is increased by changes in
mechanical loading of muscle in vivo. Journal of Cellular Biochemistry, 64, 55–56.
Sprenger, H., Jacobs, C., Nain, M., Gressner, A. M., Prinz, H., Wesemann, W., et al. (1992).
Enhanced release of cytokines, interleukin-2 receptors and neopterin after long distance run-
ning. Clinical Immunology and Immunopathology, 63, 188–195.
Springer, T. A. (1994). Traffic signals for lymphocyte recruitment and leukocyte emigration: The
multistep paradigm. Cell, 76, 301–314.
Squire, J. M. (1997). Architecture and function in the muscle sarcomere. Current Opinion in
Structural Biology, 7, 247–257.
Squire, J. M., Al-Khayat, H. A., Knupp, C., & Luther, P. K. (2005). Molecular architecture in
muscle contractile assemblies. Advances in Protein Chemistry, 71, 17–87.
Srirangan, S., & Choy, E. H. (2010). The role of interleukin 6 in the pathophysiology of rheuma-
toid arthritis. Therapeutic Advances in Musculoskeletal Disease, 2, 247–256.
St. Pierre, B. A., & Tidball, J. G. (1994). Differential response of macrophage subpopulations
to soleus muscle reloading after rat hindlimb suspension. Journal of Applied Physiology, 77,
290–297.
Starkie, R. L., Rolland, J., Angus, D. J., Anderson, M. J., & Febraio, M. A. (2001). Circulating
monocytes are not the source of elevations in plasma IL-6 and TNF-α levels after prolonged
running. American Journal of Physiology. Cell Physiology, 280, C769–C774.
Steensberg, A., Febbraio, M. A., Osada, T., Schjerling, P., Van Hall, G., Saltin, B., et al. (2001).
Interleukin-6 production in contracting human skeletal muscle is influenced by pre-exercise
muscle glycogen content. The Journal of Physiology, 537, 633–639.
Steensberg, A., Keller, C., Starkie, R. L., Osada, T., Febbraio, M. A., & Pedersen, B. K. (2002). Il-6
and TNF-alpha expression in, and release from contracting human skeletal muscle. American
Journal of Physiology-Endocrinology and Metabolism, 283, E1272–E1278.
Steensberg, A., Van Hall, G., Osada, T., Sacchetti, M., Saltin, B., & Pedersen, B. K. (2000).
Production of interleukin-6 in contracting human skeletal muscles can account for the exercise-
induced increase in plasma interleukin-6. The Journal of Physiology, 529, 237–242.
Stevens, S. S. (1971). Issues in psychophysical measurement. Psychological Review, 78, 426–450.
References 123
Stone, M. R., O’Neill, A., Catino, D., & Bloch, R. J. (2005). Specific interaction of the actin-
binding domain of dystrophin with intermediate filaments contraining keratin 19. Molecular
Biology of the Cell, 16, 4280–4293.
Stouthard, J. M. L., Romjin, J. A., Van Der Poll, T., Endert, E., Klein, S., Bakker, P. J. M., et al.
(1995). Endocrinologic and metabolic effects of interleukin-6 in humans. American Journal of
Physiology-Endocrinology and Metabolism, 268, E813–E819.
Streetz, K. L., Wustefeld, T., Klein, C., Manns, M. P., & Trautwein, C. (2001). Mediators of inflam-
mation and acute phase response in the liver. Cellular and Molecular Biology (Noisy-le-Grand,
France), 47, 661–673.
Stromer, M. H. (1995). Immunocytochemistry of the muscle cell cytoskeleton. Microscopy
Research and Technique, 31, 95–105.
Stromer, M. H. (1998). The cytoskeleton in skeletal, cardiac and smooth muscle cells. Histology
and Histopathology, 13, 283–291.
Stromer, M. H., & Goll, D. E. (1972). Studies on purified α-actinin. Journal of Molecular Biology,
67, 489–494.
Subramanian, A., & Schilling, T. F. (2015). Tendon development and musculoskeletal assembly:
Emerging role for the extracellular matrix. Development, 142, 4191–4204.
Summers, A. P., & Koob, T. J. (2002). The evolution of tendon-morphology and material proper-
ties. Comparative Biochemistry and Physiology, 133, 1159–1170.
Summers, C., Rankin, S. M., Condliffe, A. M., Singh, N., Michael Peters, A., & Chilvers, E. R.
(2010). Neutrophil kinetics in health and disease. Trends in Immunology, 31, 318–324.
Sundd, P., Pospieszalska, M. K., & Ley, K. (2013). Neutrophil rolling at high shear: Flattening,
catch bond behavior, tethers and slings. Molecular Immunology, 55, 59–69.
Suzuki, K., Hata, S., Kawabata, Y., & Sorimachi, H. (2004). Structure, activation, and biology of
calpain. Diabetes, 53, S12–S18.
Szczesna, D., & Potter, J. D. (2002). The role of troponin in the Ca2+ - Regulation of skeletal
muscle contraction. Results and Problems in Cell Differentiation, 36, 171–190.
Takahasi, K. (Ed.). (1990). Calpain substrate specificity. Boca Ratan, FL: CRC Press.
Takahasi, M., Wand, S. R., Marchuk, L. L., Frank, C. B., & Lieber, R. L. (2010). Asynchronous
muscle tendon adaptation after surgical tensioning procedures. JBJS, 92, 664–674.
Talag, T. S. (1973). Residual muscular soreness as influenced by concentric, eccentric and static
contractions. Research Quarterly, 44, 458–469.
Tate, C., Hyek, M. F., & Taffet, G. E. (1991). The role of calcium in the energetics of contracting
skeletal muscle. Sports Medicine, 12, 208–217.
Taylor, D. C., Dalton, J. D., Seaber, A. V., & Garrett, W. E. (1990). Viscoelastic properties of
muscle-tendon units. The biomechanical effects of stretching. American Journal of Sports
Medicine, 18, 300–309.
Thacker, S., Gilchrist, J. S., Stroup, D., & Kimsey Jr., C. (2004). The impact of stretching on sports
injury risk: A systematic review of the literature. Medicine and Science in Sports and Exercise,
36, 371–378.
The Free Dictionary. (2016). Autocrine [Online]. Retrieved May 31, from http://medical-
dictionary.thefreedictionary.com/autocrine
Thomas, L. (1972). Notes of a biology-watcher: Germs. The New England Journal of Medicine,
287, 535–555.
Tidball, J. G. (1983). The geometry of actin filament-membrane associations can modify adhesive
strength of myotendinous junction. Cell Motility, 3, 439–447.
Tidball, J. G. (1984). Myotendinous junction: Morphological changes and mechanical failure asso-
ciated with muscle cell atrophy. Experimental and Molecular Pathology, 40, 1–12.
Tidball, J. G. (1991). Force transmission across muscle cell membranes. Journal of Biomechanics,
24, 43–52.
Tidball, J. G. (2002). Interactions between muscle and the immune system during modified muscu-
loskeletal loading. Clinical Orthopaedics and Related Research, 403S, S100–S109.
Tidball, J. G. (2005). Inflammatory processes in muscle injury and repair. American Journal of
Physiology. Regulatory, Integrative and Comparative Physiology, 288, R345–R353.
Tidball, J. G., Burchenko, E., & Frenette, J. (1999). Macrophage invasion does not contribute to
muscle membrane injury during inflammation. Journal of Leukocyte Biology, 65, 492–498.
Tidball, J. G., O’Halloran, T., & Burridge, K. (1986). Talin at myotendinous junctions. JCB, 103,
1465–1472.
Tidball, J. G., & Quan, D. M. (1992). Modifications in myotendinous junction structure following
denervation. Acta Neropathologica, 84, 135–140.
Tidball, J. G., & Villalta, S. A. (2010). Regulatory interactions between muscle and the immune
system during muscle regeneration. American Journal of Physiology-Regulatory, Integrative
and Comparative Physiology, 298, R1173–R1187.
Tiku, K., Tiku, M. L., & Skosey, J. (1986). Interleukin 1 production by human polymorphonuclear
neutrophils. Journal of Immunology, 136, 3677–3685.
Tilg, H., Dinaello, C. A., & Meir, J. W. (1997). IL-6 and APPs: Anti-inflammatory and immuno-
suppressive mediators. Immunology Today, 18, 428–432.
Tilg, H., Trehu, E., Atkins, M. B., Dinaello, C. A., & Meir, J. W. (1994). Interleukin-6 (IL-6)
an anti-inflammatory cytokine: Induction of circulating IL-1 receptor anatagonist and soluble
necrosis factor receptor p55. Blood, 83, 113–118.
Torgan, C. E., & Daniels, M. P. (2000). Calcineurin localization in skeletal muscle offers insights
into potential new targets. The Journal of Histochemistry and Cytochemistry, 54, 119–128.
Torgan, C. E., & Daniels, M. P. (2001). Regulation of myosin heavy chain expression during rat
skeletal muscle development in vivo. Molecular Biology of the Cell, 12, 1499–1508.
Toumi, H., & Best, T. M. (2003). The inflammatory response: Friend or enemy for muscle injury?
BJSM, 37, 284–286.
Trinick, J. (1996). Cytoskeleton: Titin has a scaffold and spring. Current Biology, 6, 258–260.
Trombitas, K., Greaser, M. L., Labeit, S., Jin, J.-P., Kellermayer, M., Helmes, M., et al. (1998).
Titin extensibility in situ: Entropic elasticity of permanently folded and permanently unfolded
molecular segments. The Journal of Cell Biology, 140, 853–859.
Trotter, J. A. (1993). Functional morphology of force transmission in skeletal muscle. Acta
Anatomica, 146, 205–222.
Trotter, J. A., Corbett, K., & Avner, B. P. (1981). Structure and function of the murine muscle-
tendon junction. The Anatomical Record, 201, 293–302.
Trotter, J. A., Eberhard, S., & Samora, A. (1983). Structural connections of the muscle-tendon
junction. Cell Motility, 3, 431–438.
Trotter, J. A., Hsi, K., Samora, A., & Wofsy, C. (1985). A morphometric analysis of the muscle-
Trotter, J. A., Samora, A., & Baca, J. (1985). Three-dimensional structure of the murine muscle-
Trybus, K. M. (1994). Role of myosin light chains. Journal of Muscle Research and Cell Motility,
15, 587–594.
Tsigos, C., Papanicolaou, D. A., Kyrou, I., Defensor, R., Mitsiadis, C. S., & Chrousos, G. P.
(1997). Dose-dependent effects of recombinant human interleukin-6 on glucose regulation.
The Journal of Clinical Endocrinology and Metabolism, 82, 4167–4170.
Tskhovrebova, L., Houmeida, A., & Trinick, J. (2006). Can the passive elasticity of muscle
be explained directly from the mechanics of individual titin molecules? Journal of Muscle
Research and Cell Motility, 26, 285–289.
Tskhovrebova, L., & Trinick, J. (2001). Flexibility and extensibility in the titin molecule: Analysis
of electron microscope data. Journal of Molecular Biology, 310, 755–771.
Tskhovrebova, L., & Trinick, J. (2003). Titin: Properties and family relationships. Nature Reviews.
Molecular Cell Biology, 4, 1405–1414.
References 125
Tskhovrebova, L., Walker, M. L., Gunter Grossman, J., Nasir Khan, G., Baron, A., & Trinick,
J. (2010). Shape and flexibility in the Titin 11-domain super repeat. Journal of Molecular
Biology, 397, 1092–1105.
Tu, M. K., Levin, J. B., Hamilton, A. N., & Borodinsky, L. N. (2016). Calcium signaling in skeletal
muscle development, maintenance and regulation. Cell Calcium, 59, 91–97.
Uda, S., & Kuroda, S. (2016). Analysis of cellular signal transduction from an information theo-
retic approach. Seminars in Cell & Developmental Biology, 51, 24–31.
Ullum, H., Haahr, P. M., Diamant, M., Palmo, J., Halkjaer-Kristensen, J., & Pedersen, B. K. (1994).
Bicycle exercise enhances plasma IL-6 but does not change IL-1α, IL-1β, IL-6 or TNF-α pre-
mRNA in BMNC. Journal of Applied Physiology, 77, 93–97.
Unick, J., Kieffer, S. H., Cheesman, W., & Feeney, A. (2005). The acute effects of static and
ballistic stretching on vertical jump performance in trained women. Journal of Strength and
Valdivia, M., Vega-Macaya, F., & Olguin, P. (2017). Mechanical control of myotendinous junction
formation and tendon differentiation during development. Frontiers in Cell and Development
Biology, 5, 1–8.
Van Der Flier, A., & Sonnenberg, A. (2001). Function and interactions of integrins. Cell and Tissue
Research, 305, 285–298.
Van Der Ven, P. F., Bartsch, J. W., & Gautel, M. (2000). A Functional knock-out of titin results in
defective myofibril assembly. JCS, 113, 1405–1414.
Van Regenmortel, M. H. V. (2004). Reductionism and complexity in molecular biology. EMBO
Reports, 5, 1016–1020.
Vandenburgh, H., Hatfaludy, S., Sohar, I., & Shansky, J. (1990). Stretch induced prostaglandins
and protein turnover in cultured skeletal muscle. The American Journal of Physiology, 259,
C232–C240.
Vandenburgh, H. H., Hatfaludy, S., Karlisch, P., & Shansky, J. (1991). Mechanically induced alter-
ations in cultured skeletal muscle growth. Journal of Biomechanics, 24, 91–99.
Veskler, B. A. (Ed.). (2005). Progress in Immunology Research. New York: Nova Science
Publishers.
Vestweber, D. (2007). Adhesion and signaling molecules controlling the transmigration of leuko-
cytes through endothelium. Immunological Reviews, 218, 178–196.
Vgontzas, A. N., Papanicolaou, D. A., Bixler, E. O., Lotsikas, A., Zachman, K., Kales, A., et al.
(1999). Circadian interleukin-6 secretion and quantity and depth of sleep. The Journal of
Clinical Endocrinology and Metabolism, 84, 2603–2607.
Vigushin, D. M., Pepys, M. B., & Hawkins, P. N. (1993). Metabolic and scintigraphic studies
of radioiodinated human C-reactive protein in health and disease. The Journal of Clinical
Investigation, 91, 1351–1357.
Vihko, V., Rantamaki, J., & Salminen, A. (1978). Exhaustive physical exercise and acid hydralase
activity in mouse skeletal muscle. Histochemistry, 57, 237–249.
Viidik, A. (1969). Tensile strength properties of Achilles tendon systems in trained and untrained
rabbits. Acta Orthopaedica Scandinavica, 40, 261–272.
Vikstrom, K. L., Seiler, S. H., Sohn, R. L., Strauss, M., Weiss, A., Welikson, R. E., et al. (1997).
The vertebrate myosin heavy chain: Genetics and assembly properties. Cell Structure and
Function, 22, 123–129.
Voermans, N. C., Bonnemann, C. G., Huijing, P. A., Hamel, B. C., Van Kuppevelt, T. H., De Haan,
A., et al. (2008). Clinical and molecular overlap between myopathies and inherited connective
tissue diseases. Neuromuscular Disorders, 18, 843–856.
Volk, T. (1995). Metapatterns - Across space, time, and mind. New York: Columbia University
Press.
Von Bertalanffy, L. (1968). General system theory: Foundations, development, applications.
New York: George Braziller.
Von Der Ecken, J., Muller, M., Lehman, W., Manstein, D. J., Penczek, P. A., & Raunser, S. (2015).
Structure of the F-actin-tropomyosin complex. Nature, 519, 114–117.
Von Vietinghoff, S., & Ley, K. (2008). Homeostatic regulation of blood neutrophil counts. Journal
Voss, D. (1967). Proprioceptive neuromuscular facilitation. American Journal of Physical
Medicine, 46, 838–898.
Wahl, S. M., Hunt, D. A., Wakefield, L. M., Mccartney-Francis, N., Wahl, L. M., Roberts, A. B.,
et al. (1987). Transforming growth factor type beta induces monocyte chemotaxis and growth
factor production. Proceedings of the National Academy of Sciences of the USA, 84, 5788–5792.
Wainwright, S. A. (1988). Axis and Circumference - The cylindrical shape of plants and animals.
Cambridge, MA: Harvard University Press.
Wakabayashi, T. (2015). Mechanism of the calcium-regulation of muscle contraction - In pursuit
of its structural basis. Proceedings of the Japan Academy. Series B, Physical and Biological
Sciences, 91, 321–350.
Wallin, D., Ekblom, B., Grahn, R., & Nordenborg, T. (1985). Improvement of muscle flexibility-a
comparison between two techniques. The American Journal of Sports Medicine, 13, 263–268.
Walsh, L. D., Allen, T. G., Gandevia, S. C., & Proske, U. (2006). Effect of eccentric exercise on
position sense at the human forearm in different postures. Journal of Applied Physiology, 100,
1109–1116.
Wang, J. H., Yang, G., Li, Z., & Shen, W. (2004). Fibroblast responses to cyclic mechanical
stretching depend on cell orientation to the stretching direction. Journal of Biomechanics, 37,
573–576.
Wang, J. H.-C., Thampatty, B. P., Lin, J.-S., & Im, H. J. (2007). Mechanoregulation of gene expres-
sion in fibroblasts. Gene, 391, 1–15.
Wang, K., Mccarter, R., Wright, J., Beverly, J., & Ramirez-Mitchell, R. (1993). Viscoelasticity of
the sarcomere matrix of skeletal muscles-the titin-myosin composite filament is a dual-stage
molecular spring. Biophysical Journal, 64, 1161–1177.
Wang, K., & Ramirez-Mitchell, R. (1983). A network of transverse and longitudinal intermediate
filaments is associated with sarcomeres of adult vertebrate skeletal muscle. The Journal of Cell
Biology, 92, 562–570.
Wang, K., & Wright, J. (1988). Architecture of the sarcomere matrix of skeletal muscle:
Immunoelectron microscopic evidence that suggests a set of parallel inextensible nebulin fila-
ments anchored at the Z line. JCB, 107, 2199–2212.
Wang, K., Wright, J., & Ramirez-Mitchell, R. (1985). Architecture of the titin/nebulin containing
cytoskeletal lattice of the striated muscle sarcomere-evidence of elastic and inelastic domains
of the bipolar filaments. Biophysical Journal, 47, 349a.
Ward, P. A. (Ed.). (2010). Part I. The inflammatory response - An overview. Cambridge, UK:
Cambridge University Press.
Warren, G. L., Lowe, D. A., & Armstrong, R. B. (1993). Mechanical factors in the initiation of
eccentric contraction-induced injury in rat soleus muscle. The Journal of Physiology, 464,
457–475.
Watson, P. A. (1991). Function follows form: Generation of intracellular signals by cell deforma-
tion. The FASEB Journal, 5, 2013–2019.
Weerapong, P., Hume, P., & Kolt, G. (2004). Stretching: Mechanisms and benefits for sport perfor-
mance and injury prevention. The Physical Therapy Review, 9, 189–206.
Weller, A., Isenmann, S., & Vestweber, D. (1992). Cloning of the mouse endothelial selectins-
expression of both E- and P- selectin is inducible by tumor necrosis factor α. Proceedings of
the National Academy of Sciences of the USA, 267, 15176–15183.
Wessel, J., & Wan, A. (1994). Effect of stretching on the intensity of delayed-onset muscle sore-
ness. Clinical Journal of Sport Medicine, 4, 83–87.
West, J. B. (2014). Galen and the beginnings of western philosophy. American Journal of
Physiology. Lung Cellular and Molecular Physiology, 307, L121–L128.
Westerhoff, H. V., & Palsson, B. O. (2004). The evolution of molecular biology into systems biol-
ogy. Nature Biotechnology, 22, 1249–1252.
References 127
Wette, S. G., Smith, H. K., Lamb, G. D., & Murphy, R. M. (2017). Characterization of mus-
cle ankyrin repeat proteins in human skeletal muscle. American Journal of Physiology. Cell
Physiology, 313, C327–C339.
Whicher, J. T., & Westacott, C. I. (Eds.). (1992). The acute phase response. London: Kluwer
Academic.
White, S. P., Cohen, C., & Phillips Jr., G. N. (1987). Structure of co-crystals of tropomyosin and
troponin. Nature, 325, 745–830.
Wickiewicz, T. L., Roy, R. R., Powell, P. L., & Edgerton, V. R. (1983). Muscle architecture of the
human lower limb. Clinical Orthopaedics and Related Research, 179, 275–283.
Wickiewicz, T. L., Roy, R. R., Powell, P. L., Petrine, J. J., & Edgerton, V. R. (1984). Muscle
architecture and force velocity relationships in humans. Journal of Applied Physiology, 57,
435–443.
Willems, J., Joniau, M., Cinque, S., & Van Damme, J. (1989). Human granulocyte chemotac-
tic peptide (IL-8) as a specific neutrophil degranulator: Comparison with other monokines.
Williams, P. E., & Goldspink, G. (1971). Longitudinal growth of striated muscle fibers. Journal of
Cell Science, 9, 751–767.
Williams, P. E., & Goldspink, G. (1978a). Changes in sarcomere length and physiological proper-
ties in immobilized muscle. Journal of Anatomy, 127, 459–468.
Williams, P. E., & Goldspink, G. (1978b). The effect of immobilization on the longitudinal growth
of striated muscle fibers. Journal of Anatomy, 116, 45–55.
Wilson, G., Elliot, B., & Wood, G. (1992). Stretch shorten cycle performance enhancement through
flexibility training. Medicine and Science in Sports and Exercise, 24, 116–123.
Wilson, G., Wood, G., & Elliot, B. (1991). The relationship between stiffness of the musculature and
static flexibility: An alternative explanation for the occurrence of muscular injury. International
Journal of Sports Medicine, 12, 403–407.
Winchester, J. B., Nelson, A. G., Landin, D., Young, M. A., & Schexnayder, I. C. (2008). Static
stretching impairs sprint performance in collegiate track and field athletes. Journal of Strength
and Conditioning Research, 22, 13–19.
Witt, C. C., Burkart, C., Labeit, D., Mcnabb, M., Wu, Y., & Granzier, H. (2006). Nebulin regu-
lates thin filament length, contractility, and Z-disk structure in vivo. The EMBO Journal, 25,
3843–3855.
Witvrouw, E., Mahieu, N., Roosen, P., & Mcnair, P. (2007). The role of stretching in tendon inju-
ries. British Journal of Sports Medicine, 41, 224–226.
Woo, L.-Y. S., & Buckwalter, J. A. (1988). Injury and repair of the musculoskeletal soft tissues.
Park Ridge, IL: American Academy of Orthopaedic Surgeons.
Woo, S. Y., Maynard, J., Butler, D., Lyon, R., Torzilli, P., Akeson, W., et al. (1988). Ligament,
Tendon, and Joint Capsule Insertions to Bone. Savannah, GA: American Academy of
Orthopaedic Surgeons.
Wren, T. A., Beaupre, G. S., & Carter, D. R. (2000). Mechanobiology of tendon adaptation to
compressive loading through fibrocartilage metaplasia. Journal of Rehabilitation Research and
Development, 37, 135–143.
Wu, M., Fannin, J., Rice, K. M., Wang, B., & Blough, E. R. (2011). Effect of aging on cellular
mechanotransduction. Ageing Research Reviews, 10, 1–15.
Wynn, T. A. (2008). Cellular and molecular mechanisms of fibrosis. The Journal of Pathology,
214, 199–210.
Wynn, T. A., & Vannella, K. M. (2016). Macrophages in tissue repair, regeneration, and fibrosis.
Immunity, 44, 450–462.
Xia, S., & Kanchanawong, P. (2017). Nanoscale mechanobiology of cell adhesions. Seminars in
Cell & Developmental Biology, 71, 53–67.
Xu, S., Gu, J., Rhodes, T., Belknap, B., Rosenbaum, G., Offer, G., et al. (1999). The M.ADP.
Pi state is required for helical order in the thick filaments of skeletal muscles. Biophysical
Journal, 77, 2665–2676.
Yamashita, A., Maeda, K., & Maeda, Y. (2003). Crystal structure of CapZ: Structural basis for actin
filament barbed end capping. The EMBO Journal, 22, 1529–1538.
Yanagishita, M. (1993). Function of proteoglycans in the extracellular matrix. Acta Pathologica
Japonica, 43, 283–293.
Yang, J., Zhang, L., Yu, C., Yang, X.-F., & Wang, H. (2014). Monocyte and macrophage differenti-
ation: Circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomarker
Research, 2, 1–9.
Yavropoulou, M. P., & Yovos, J. G. (2016). The molecular basis of bone mechanotransduction.
Journal of Musculoskeletal & Neuronal Interactions, 16, 221–236.
Yeung, E. W., Belnave, C. D., Ballard, H. J., Bourreau, J.-P., & Allen, D. G. (2002). Development
of T-tubular vacuoles in eccentrically damaged mouse muscle fibres. The Journal of Physiology,
540, 581–592.
Yoshida, Y., & Tanaka, T. (2014). Interleukin 6 and rheumatoid arthritis. BioMed Research
International, 2014, 1–12.
Young, P., Ehler, E., & Gautel, M. (2001). Obscurin, a giant sarcomeric Rho guanine nucleotide
exchange factor protein involved in sarcomere assembly. JCB, 154, 123–136.
Young, W., & Behm, D. (2002). Should static stretching be used during a warm-up or for strength
and power activities? Strength & Conditioning Journal, 24, 33–37.
Young, W., Elias, G., & Power, J. (2006). Effects of static stretching volume and intensity on
planter flexor explosive force production and range of motion. The Journal of Sports Medicine
and Physical Fitness, 46, 403–411.
Young, W. B., & Behm, D. G. (2003). Effects of running, static stretching and practice jumps on
explosive force production and jumping performance. The Journal of Sports Medicine and
Physical Fitness, 43, 21–27.
Zhang, J.-M., & An, J. (2007). Cytokines, inflammation and pain. International Anesthesiology
Clinics, 45, 27–37.
Zhou, Q., Chu, P.-H., Huang, C., Cheng, C.-F., Martone, M. E., Knoll, G., et al. (2001). Ablation
of cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy.
JCB, 155, 605–612.
Zimkowska, M., Duchesnay, A., Dragun, P., Oberbek, A., Moraczewski, J., & Martelly, I. (2009).
Immunoneutralization of TGFβ1 improves skeletal muscle regeneration: Effects on myoblast
differentiation and glycosaminoglycan content. International Journal of Cell Biology, 2009,
1–16.
Zot, A. S., & Potter, J. D. (1987). Structural aspect of troponin-tropomyosin regulation of skeletal
muscle contraction. Annual Review of Biophysics and Biophysical Chemistry, 16, 535–559.
Further Readings
A. Molecular Structure of Muscle
Barczyk, M., Carracedo, S., & Gulberg, D. (2010). Integrins. Cell and Tissue Research, 339,
269–280.
Burkin, D. J., & Kaufman, S. J. (1999). The a7b1 integrin in muscle development and disease. Cell
and Tissue Research, 296, 183–190.
Campbell, I. D., & Humphries, M. J. (2011). Integrin structure, activation, and interactions. Cold
Spring Harbor Perspectives in Biology, 2011, 1–14.
Clark, K. A., Mcelhinny, A. S., Beckerle, M. C., & Gregorio, C. C. (2002). Striated muscle cyto-
architecture: An intricate web of form and function. Annual Review of Cell and Developmental
Biology, 18, 637–706.
Craig, R. W., & Padron, R. (2004). Molecular structure of the sarcomere. In A. C. Engel &
C. Franzini-Armstrong (Eds.), Myology (3rd ed.). New York: McGraw-Hill.
References 129
Geeves, M. A., & Holmes, K. C. (2005). The molecular mechanism of muscle contraction.
Advances in Protein Chemistry, 71, 161–193.
Green, L. J., Mould, P., & Humphries, M. J. (1998). The integrin b subunit. The International
Journal of Biochemistry & Cell Biology, 30, 179–184.
Holmberg, J., & Durbeej, M. (2013). Laminin-211 in skeletal muscle function. Cell Adhesion &
Migration, 7, 111–121.
Kontrogianni-Konstantopoulos, A., Ackermann, M. A., Bowman, A. L., Yap, S. V., & Bloch, R. J.
(2009). Muscle giants: Molecular scaffolds in sarcomeregenesis. Physiological Reviews, 89,
1217–1267.
Mayer, U. (2003). Integrins: Redundant or important players in skeletal muscle. JBC, 278,
14587–14590.
Plow, E. F., Haas, T. A., Zhang, L., Loftus, J., & Smith, J. W. (2000). Ligand binding to integrins.
JBC, 275, 21785–21788.
Squire, J. M., Al-Khayat, H. A., Knupp, C., & Luther, P. K. (2005). Molecular architecture in
muscle contractile assemblies. Advances in Protein Chemistry, 71, 17–87.
B. Myotendon Unit
Charvet, B., Ruggiero, F., & Le Guellec, D. (2012). The development of the myotendinous junc-
tion. A review. Muscles, Ligaments, and Tendons Journal, 2, 53–63.
Ricard-Blum, S. (2011). The collagen family. Cold Spring Harbor Perspectives in Biology, 3,
a004978.
C. Mechanotransduction
Humphrey, J. D., Dufresne, E. R., & Schwartz, M. A. (2014). Mechanotransduction and extracel-
lular matrix homeostasis. Nature Reviews. Molecular Cell Biology, 15, 802–812.
D. Rolling, Adhesion Transmigration
Ley, K., Laudanna, C., Cybulsky, M. I., & Noursharhg, S. (2007). Getting to the site of inflamma-
tion: The leukocyte adhesion cascade update. Nature Reviews Immunology, 7, 678–689.
Muller, W. A. (2013). Getting leukocytes to the site of inflammation. Veterinary Pathology, 50,
7–22.
Sundd, P., Pospieszalska, M. K., & Ley, K. (2013). Neutrophil rolling at high shear: Flattening,
catch bond behavior, tethers and slings. Molecular Immunology, 55, 59–69.
Chapter 3
Study One: Acute Inflammatory Response
to Stretching
Introduction
Traditionally stretching exercises have been advocated by sports coaches and

medical professionals as a means for performance enhancement and injury preven-
tion (Taylor, Dalton, Seaber, & Garrett, 1990). The ability of connective and muscu-
lar tissues to change their architecture in response to stretching is important for
proper function, repair, and performance, and is dependent upon the active and pas-
sive tension of the muscle, the MTU, the muscle spindles, and the Golgi tendon
organs (GTO) (Apostolopoulos, Metsios, Flouris, Koutedakis, & Wyon, 2015;
Guissard & Duchateau, 2006; Nikolaou, Macdonald, Glisson, Seaber, & Garrett Jr,
1987). Active tension is the interaction of the actin and myosin filaments of muscle,
with passive referring to the elongation of the connective tissue beyond its resting
length (Knudson, 2006). Both define the length-dependent properties of muscle and
are strongly influenced by stretching, specifically the interaction of the tissue and
the nature of the training stimulus (Knudson, 2006). In other words, when muscle is
stretched with use of various stretching techniques, these techniques account for
changes in the active and passive tension of muscle, thereby improving the ROM
about a joint (Knudson, 2006).
Stretching, a time-dependent activity, relies on the parameters of training: inten-
sity, duration, and frequency (Marschall, 1999; Mujika et al., 1995). Of these three,
intensity, defined as the magnitude and rate of force applied to a joint during stretch-
ing (Jacobs & Sciacia, 2011; Mcclure, Blackburn, & Dusold, 1994), may be of
greater importance, directly influencing ROM (Marschall, 1999). Stretching inten-
sity may play a primary role in determining stretching outcomes; for if the applied
force is too high, it may injure the tissue, triggering an inflammatory response
(Jacobs & Sciacia, 2011).1 A fourth parameter, body position, the position assumed
during stretching, may influence the magnitude of the force generated prior to and
Parts of this chapter appear in Apostolopoulos et al. (2015).

1

132 3 Study One: Acute Inflammatory Response to Stretching
during stretching, affecting the response of the muscle and tendon tissue
(Apostolopoulos et al., 2015). A study comparing a standing to a supine hamstring
stretch observed that the latter isolated the hamstring better, was more comfortable,
but more importantly facilitated a better relaxation response (Abdel-Aziem, Draz,
Mosaad, & Abdelraou, 2013). The principle of stability, balance, and control (SBC)
summarises the importance of body position and stretching intensity suggesting that
one is better able to control the amount of stress imparted on the muscle, thereby
limiting any pain and discomfort, ensuring a better adaptation of the muscle to a
mechanical load (Apostolopoulos, 2010). The importance of stretch intensity (level
of pain and discomfort), and the influence of body position (relaxed or tense mus-
cle), are made clearer when considering the definition of pain as suggested by the
International Association for the Study of Pain. Pain is “an unpleasant sensory and
emotional experience associated with actual or potential tissue damage, or described
in terms of such damage” (Merskey & Bogduk, 1994). Acute muscle damage caused
by overloading and/or training injuries initiates a sequence of cellular responses
triggering an inflammatory response (Tidball, 1995).
A previous study comparing static to ballistic stretching revealed that static
stretching produced higher levels of DOMS, and was associated with the release of
creatine kinase, suggesting muscle damage. Muscle damage itself is associated with
the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-a) (Chatzinikolaou
et al., 2010; Chiu et al., 2013; Cunniffe et al., 2010, 2011; Lund, Hurst, Tyrell, &
Thompson, 2011). Of the pro-inflammatory cytokines, IL-6 is directly responsible
for the release of C-reactive protein, a blood test marker for inflammation produced
by the liver. C-reactive protein is classified as an acute phase reactant, meaning that
its levels rise in response to inflammation, and is considered a principal downstream
mediator of the acute phase response (Akira, Hirano, Taga, & Kishimoto, 1990;
Gabay, 2006; Kilicarslan, Uysal, & Roach, 2013; Pradhan, Manson, Rifai, Buring,
& Ridker, 2001; Ramadori, Van Damme, Rieder, & Meyer Zum Buschenfelde,
1988).
Therefore, the aim of this study was to investigate whether acute high-intensity
passive static stretching (IS) causes an inflammatory response. We hypothesised
that changes in the concentrations of the C-reactive protein, as well as the pro-
inflammatory cytokines (IL-1β, IL-6, and TNF-α) should be observed between IS
and control interventions.
Methods
Participants
Twelve recreationally active male athletes (age, 29 ± 4.33 years; mass, 79.3 ± 8.78 kg;

height, 1.76 ± 0.06 m) were recruited for the study. Each participant read and signed
an informed consent form, and answered a physical activity readiness questionnaire
Methods 133
(PARQ) and blood analysis questionnaire, screening for confounding factors that
may influence the outcomes (e.g. smoking, injury, chronic disease). Ethical approval
for the study was granted by the University of Wolverhampton’s ethics committee,
with the rights of each participant being protected. Participants were instructed to
avoid any unconventional strenuous activity prior to, as well as post-stretching
intervention, and during pre-24 h blood collections.
Power Calculations
C-reactive protein was selected as the primary endpoint for its importance as an
inflammatory biomarker as it has been previously assessed pre- and postexercise in
stretching (Frey et al., 1994). Assuming a detectable difference of 2 mg/L with
2 mg/L standard deviation, and an 80% power with an alpha level of 5%, a sample
size of 11 participants was required; to allow for potential dropouts, we recruited 12
participants (nQuery Advisor v 6.0, Statistical Solutions, MA, USA).
Procedures
Participants were randomised into either an IS or control group, using a computer-

randomised programme (www.sealedenvelope.com) (Fig. 3.1), and were tested in a
controlled setting at the University of Wolverhampton’s physiology laboratory.
During their first session, they were familiarised with their respective activity
(stretch intervention or control), with measurements taken for height and body
mass. All participants acted as their own control.
Measurement of hsCRP was utilised for quantifying C-reactive protein. Studies
conducted with apparently healthy individuals require hsCRP methods (Roberts
et al., 2001), allowing for detection of C-reactive protein levels an order of magni-
tude lower than traditional assays. This enables measurement of low-level eleva-
tions in C-reactive protein, where there is a local, low-grade inflammatory
component (Pearle et al., 2007). This refers to a component describing conditions
(i.e. type 2 diabetes and atherosclerosis) typically exhibiting a two- to threefold
increase in systemic concentrations of C-reactive protein, IL-1β, TNF-α, and IL-6
(Petersen & Pedersen, 2005). With clearance of C-reactive protein from plasma
having a biological half-life of 19–20 h, falling by up to 50% per day afterwards, the
week between conditions (IS and control) ensured a full clearout of any potential
effects (Marino & Giotta, 2008; Pepys & Hirschfield, 2003; Vigushin, Pepys, &
Hawkins, 1993). This clearance is independent of physiological and pathophysio-
logical circumstance(s) (Marino & Giotta, 2008; Pepys & Hirschfield, 2003;
Vigushin et al., 1993).
Fig. 3.1 Methodology schematic of intervention(s) (Published with kind permission of Copyright
© Nikos C. Apostolopoulos 2018. All Rights Reserved)
High-Intensity Passive Static Stretching (IS) Protocol
Participants were asked to rest in the supine position on a floor mat for 10 min with
support provided under the knees. After the rest period, a trained therapist began
stretching the muscles of the lower extremity (hamstrings, gluteals, and quadriceps)
for each participant. The therapist was instructed to maintain constant pressure on
the muscle groups throughout the duration of each stretch (60 s). With perception of
discomfort or pain varying amongst participants, a numerical rating scale adopted
from McCaffery et al. (Mccaffery & Beebe, 1989) was utilised to standardise the
intensity of the stretch. This scale was anchored at zero (0) “no pain” to ten (10)
“worst pain possible”. With participants required to experience pain and discomfort
during each stretch amid the stretching intervention, verbal encouragement was pro-
vided to maintain a level equivalent to an eight out of ten on this scale (discomfort
with slight pain). All participants were stretched for three sets, with each set
Methods 135
consisting of stretching the right hamstring and gluteal muscles in the supine
position followed by both the right and left quadriceps muscles in the prone, pro-
ceeding with the left gluteal and hamstring muscles, completing one set. With no
rest between sets, the total time for the completion of three sets was approximately
18 min. Blood samples were collected pre-, immediately post, and at 24 h post.
Control Intervention
Similar to the stretching intervention, participants rested on a floor mat in the supine
position with support provided under the knees for 10 min. This position was main-
tained for a further 18 min, mimicking the time allotted for the high-intensity pas-
sive static stretching intervention, with blood samples collected pre-, immediately
post, and 24 h post.
Biomarkers
Approximately 5 mL of each participant’s blood was drawn from the cephalic vein
of the arm of choice by a certified phlebotomist pre-, immediately post-, and 24 h
post-IS and control sessions. Serum hsCRP (eBioscience—BMS8288FF), IL-1β
(eBioscience—BMS8224FF), TNF-α (eBioscience—BMS8223FF), and IL-6
(eBioscience—BMS8213FF) were measured by means of a FlowCytomix Simplex
Kit (San Diego, CA, USA) with a Beckman FC500 flow cytometer. The flow cytom-
eter, used routinely in both research and clinical labs to study normal and abnormal
cell function and structure, as well as diagnose and monitor human disease and
responses to therapy, measures the light scatter and fluorescence emission proper-
ties of individual cells in each sample (Aghaeepour et al., 2013). It is well adapted
to measure soluble analytes (a compound of interest in an analytical procedure),
such as variations in cytokines and C-reactive protein in the sera and plasma sam-
ples of both healthy and non-healthy individuals (O’Hara et al., 2011; Prunet et al.,
2006). Its advantage is that it allows for detection of several functional characteristic
of a cell simultaneously (Green et al., 2011; O’Hara et al., 2011).
Statistical Analysis
Pre-analysis screening using the Kolmogorov-Smirnov test was employed to estab-

lish distribution of all the variables. Accordingly, LN transformation was used to
overcome skewness and kurtosis in the dependent variables of interest (hsCRP,
IL-1β, TNF-α, and IL-6) in relation to the independent variables (IS and control). A
repeated measures analysis of variance (RMANOVA) evaluated the changes in
the dependent variables over time, intervention, and time, for all the dependent
variables, with the least significant difference (LSD) post hoc test evaluating any
differences based on an observed main effect. With the level of significance set at
p < 0.05, all statistical analyses were performed via SPSS (version 20.0, SPSS Inc.,
USA). It should be noted that although 12 participants were recruited for the study,
there was missing data for hsCRP blood biomarkers. With regard to the control
condition, one value was missing for pre-hsCRP, two for post hsCRP, and one for
24 h post hsCRP. Complete set of data points for both pre- and post-IS exists with
one missing value recorded for 24 h post. Therefore, complete data for both condi-
tions and all time points (pre-, post, and 24 h post) existed for nine participants. In
addition, effect sizes (Cohen’s “d”) were calculated comparing in-between (pre-,
post, and 24 h post) and within groups (pre- vs. post, pre- vs. 24 h post, and post vs.
24 h post) for both the IS and control conditions.
Results
RMANOVA
All participants were involved in both interventions (IS and control), with no with-
drawals. The RMANOVA revealed that Mauchly’s test for sphericity was violated
for time and time × intervention for hsCRP (p = 0.002), X2 (2) = 12.48, p < 0.05.
With the degrees of freedom corrected using the Greenhouse-Geisser estimates of
sphericity (ε = 0.658), significant differences were detected for time (p = 0.005) and
time × intervention (p = 0.006). No significance was observed for IL-1β (time,
p = 0.277; time × intervention, p = 0.815), TNFα (time, p = 0.462; time × interven-
tion, p = 0.435), and IL-6 (time, p = 0.274; time × intervention, p = 0.537); however,
it should be emphasised that there were insufficient data of detectable values for
these pro-inflammatory cytokines (Table 3.1).
The LSD post hoc test revealed a significant difference between pre- and 24 h
post measurements for hsCRP (p = 0.012). The mean and standard deviation LN
transformed values for pre-control and pre-IS (0.604 ± 0.155 mg/L and
0.727 ± 0.378 mg/L) compared with 24 h post (0.604 ± 0.155 mg/L and
1.343 ± 0.599 mg/L) support this increase (Table 3.1, Fig. 3.2).
Effect Sizes
Effect Sizes Between Conditions
See Table 3.2 for descriptive statistics and effect sizes for all between group and
within group comparisons. The effect size for the comparison between IS and con-
trol at baseline suggests a small elevated concentration of hsCRP in the IS interven-
tion (“d” = 0.49, 95% CI, 0.13–0.86). The effect sizes for the comparison between
Table 3.1 Mean (SD) of raw and LN transformed blood biomarkers in relation to time and
intervention(s)
Blood biomarkers
hsCRP (mg/L) IL-6 (mg/L) IL-1β (mg/L) TNF-α (mg/L)
Time Control IS Control IS Control IS Control IS
Pre-(raw) 0.85 1.24 5.0 × 10−3 0.61 0.21 1.02 0.04 6.52
(0.27) (1.14) (0.012) (2.01) (0.63) (2.83) (0.11) (21.53)
Pre-(LN) 0.60 0.73 5.0 × 10−3 0.19 0.12 0.31 0.04 0.42
(0.16) (0.38) (0.012) (0.61) (0.35) (0.74) (0.09) (1.29)
Immediately 0.79 1.81 7.0 × 10−3 0.61 0.29 0.88 0.05 3.41
post-(raw) (0.35) (1.87) (0.02) (2.01) (0.66) (2.16) (0.13) (11.17)
Immediately 0.57 0.91 7.0 × 10−3 0.19 0.17 0.33 0.04 0.36
post-(LN) (0.22) (0.45) (0.02) (0.61) (0.37) (0.68) (0.11) (1.09)
24 h 0.85 3.55 5.0 × 10−3 0.61 0.21 0.87 0.04 2.99
post-(raw) (0.27) (2.96)a (0.01) (2.02) (0.63) (2.31) (0.11) (9.77)
24 h post-(LN) 0.60 1.34 5.0 × 10−3 0.19 0.12 0.29 0.04 0.35
(0.16) (0.59)a (0.01) (0.61) (0.35) (0.69) (0.09) (1.05)
SD standard deviation, Ln natural logarithm, IS high-intensity passive static stretching, hsCRP
high-sensitivity C-reactive protein, IL-6 interleukin 6, IL-1β interleukin 1 beta, TNF-α tumour
necrosis factor alpha
a
Repeated measures ANOVA significantly different between conditions over time
Fig. 3.2 Change of hsCRP over time (h)

Table 3.2 Descriptive statistics and effect sizes for all between conditions and within conditions
comparisons
Cohen’s 95% CI
Mean difference (SD) “d” 95% CI (lower) (upper)
Comparisons between conditions
Pre-IS—pre-control 0.42 (0.94) 0.49 0.13 0.86
Post-IS—post-control 0.08 (1.44) 0.41 −0.26 1.07
24 h post-IS—24 h post-control 2.84 (2.74) 1.32 0.40 2.24
Comparisons within IS
Post-IS—pre-IS 0.52 (0.79) 0.37 −0.28 1.02
24 h post-IS—pre-IS 2.31 (2.40) 1.06 0.08 2.03
24 h post-IS—post-IS 1.75 (2.13) 0.74 −0.32 1.81
Comparisons within control
Post-control—pre-control −0.05 (0.34) 0.39 −0.00 0.78
24 h post-control—Pre-control −0.00 (0.00) −0.00 −0.17 0.16
24 h post-control—post-control 0.05 (0.34) −0.39 −0.78 0.002
SD standard deviation, IS high-intensity passive static stretching
Cohen’s “d” cutoff points for effect size interpretation: small effect ≥0.20, medium effect ≥0.50,
and large effect ≥0.80
IS and control immediately post-conditions suggest a small difference in hsCRP

concentrations (“d” = 0.41, 95% CI, −0.26 to 1.07), with higher concentrations in
the IS group. However, the upper 95% CI for Cohen’s “d” crossed zero (0) (i.e. the
line of no effect). Comparisons of effect sizes for between groups at 24 h post-
condition suggest a large difference (“d” = 1.32, 95% CI, 0.40–2.24), with higher
concentrations again in the high-intensity passive static stretching group.
Effect Sizes Within Conditions
The effect sizes for comparisons between pre- and post-condition measures revealed
a small increase in hsCRP in both the IS and control conditions (“d” = 0.39, 95%
CI, −0.00 to 0.78 and “d” = 0.37, CI, −0.28 to 1.02, respectively). However, the
95% CI for Cohen’s “d” contained 0 for both comparisons, which means that there
may be no effect. When comparing pre-condition values to 24 h post, we found a
large increase for hsCRP in the IS condition (“d” = 1.06, 95% CI, 0.08–2.03), but
no effect in the control condition (“d” = −0.00, 95% CI, −0.17 to 0.16), suggesting
a higher concentration is associated within the 24 h post- vs. pre-conditions for
IS. The effect sizes for comparisons of between immediately post-conditions and
24 h post-conditions revealed a moderate increasing effect on hsCRP in the high-
intensity passive static stretching condition (“d” = 0.75, 95% CI, −0.32 to 1.81) and
a small lowering effect in the control condition (“d” = −0.39, 95% CI, −0.78 to
0.002). However, the 95% CI for Cohen’s “d” contained zero (0) for both compari-
sons, which again may mean that no effect exists.
Discussion 139
Discussion
We assessed whether high-intensity passive static stretching (IS) results in acute

inflammation by specifically referring to selected inflammatory biomarkers
(hsCRP) and pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). A significant
increase in hsCRP at 24 h post-IS intervention compared to control (no stretching)
was observed. However, with regard to the pro-inflammatory cytokines IL-1β,
IL-6, and TNF-α, there were insufficient data of detectable values preventing rel-
evant statistical analyses. Based on the effect sizes, although there was slightly
higher baseline hsCRP concentrations in the IS condition, there appeared to be a
large increase in hsCRP at 24 h post for this intervention compared to control. The
effect sizes comparing each measurement time within each condition revealed an
at least small elevating effect on hsCRP (i.e. lower 95% CI = 0.08) when compar-
ing baseline values to 24 h post, but no consistent effects for any of the other within
group comparisons.
To our knowledge this is the first study to investigate the relationship between IS
and acute inflammation with reference to an inflammatory biomarker (hsCRP) and
pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in humans. Exercise pro-
duces a short-term inflammatory response (Kasapis & Thompson, 2005), with sev-
eral studies observing increases in C-reactive protein during, immediately after, as
well as 4 and 24 h post-strenuous exercise (Kim, Lee, & Kim, 2007; Siegel et al.,
2001; Taylor et al., 1987; Weight, Alexander, & Jacobs, 1991). In comparing base-
line values, Siegel et al. (2001) observed a twofold or greater increase in C-reactive
protein within 4 h, with Weight et al. and Taylor et al. observing a 2000% and 266%
increase 24 h post-strenuous exercise, respectively. In the study by Kim et al.,
hsCRP increased 23-fold in the second half of a 200 km ultramarathon, suggesting
that intense physical exercise induces an acute-phase inflammatory reaction. Similar
to these studies, a 14-fold increase in hsCRP was observed compared to baseline.
Although we are not claiming that high-intensity passive static stretching is similar
to intense strenuous activity, this delayed rise in hsCRP (24 h post) is very interest-
ing, suggesting a disruption in the myofibres. However, future research needs to
measure for plasma creatine phosphokinase, a widely used marker of muscle dam-
age during exercise, with increases related to both the intensity and duration of
exercise (Noakes, 1987).
With blood collected pre- and immediately post with no collection between
immediately post and at 24 h post intervention, the opportunity to measure the half-
lives for the pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) was missed. In
the literature, half-lives for TNF-α, IL-1β, and IL-6 have been reported to be approx-
imately 18.2 min (Oliver et al., 1993), 2.5 h (Moors & Mizel, 2000), and between 2
and 4 h (Marino & Giotta, 2008), respectively, with the release of IL-1β and TNF-α
responsible for the synthesis of IL-6 (Akira et al., 1990). A recent review on
exercise-induced muscle damage and inflammation illustrated that IL-6 increased
from 1 to 4 h following a bout of eccentric exercise (Paulsen, Mikkelsen, Raastad,
& Peake, 2012), with an increase in IL-1β occurring 2–3 h postexercise (Cannon,
Evans, Hughes, Meredith, & Dinarello, 1986). Increases in IL-6 are associated with
a delayed increase in C-reactive protein (Akira et al., 1990; Akira, Taga, &
Kishimoto, 1993; Chatzinikolaou et al., 2010; Cunniffe et al., 2010; Cunniffe et al.,
2011; Kim et al., 2007; Ramadori et al., 1988; Streetz, Wustefeld, Klein, Manns, &
Trautwein, 2001). Similarly to the aforementioned studies, a delayed increase in
hsCRP was observed suggesting the release of IL-6, a principle downstream media-
tor of the acute phase response and the subsequent synthesis of C-reactive protein,
with its synthesis enhanced synergistically by IL-1β (Mackiewicz, Speroff,
Ganapathi, & Kushner, 1991; Marnell, Mold, & Du Clos, 2005). This relationship
between IL-6 and C-reactive protein was observed in animal studies, with mice
deficient in IL-6 (IL-6−/−) unable to generate an acute phase response and to prop-
erly recover from inflammation (Kopf et al., 1994; McFarland-Mancini et al., 2010).
The observed sharp rise in hsCRP compared to baseline (1.34 mg/L) is in line with
studies suggesting the appearance of IL-6 into circulation dependent on the mass of
muscle recruited (Fallon, 2001; Febbraio & Pedersen, 2002; Taylor et al., 1987).
The sharp increase in IL-6 preceded increases in C-reactive protein following a
160 km triathlon race (50.8 mg/L) (Taylor et al., 1987) and a 6-day ultramarathon
(37.5 mg/L) (Fallon, 2001). In the present study, the hamstrings, glutes, and
quadriceps muscle were exposed to a high-intensity passive static stretch.
Therefore, it would be interesting to investigate if the stretching of a single muscle
group will elicit the same response with use of high-intensity passive static stretch-
ing exercise.
Conclusion
In summary and within the study’s limitations (i.e. timing of blood collection), the
present data demonstrated that high-intensity passive static stretching is associated
with an increase in systemic hsCRP attributed to inflammation and the inflamma-
tory response. Further research into the probable causes of inflammation will eluci-
date if this form of activity is detrimental to the health of the muscle tissue and its
proper function.
References
and Health Sciences, 2, 399–406.
Aghaeepour, N., Finak, G., The Flowcap Consortium, The Dream Consortium, Hoos, H.,
Mosmann, T. R., et al. (2013). Critical assessment of automated flow cytometry data analysis
techniques. Nature Methods, 10, 228–238.
References 141
Apostolopoulos, N. (2010). microStretching-A practical approach for recovery and regeneration.
New Studies in Athletics, 25, 81–97.
Cannon, J. G., Evans, W. J., Hughes, V. A., Meredith, C. N., & Dinarello, C. A. (1986). Physiological
mechanisms contributing to increased inteleukin-1 secretion. Journal of Applied Physiology,
61, 1869–1874.
Chatzinikolaou, A., Fatouros, I., Gourgoulis, V., Avloniti, A., Jamurtas, A. Z., Nikolaidis, M. G.,
et al. (2010). Time course of changes in performance and inflammatory responses after acute
plyometric exercise. Journal of Strength and Conditioning Research, 24, 1389–1398.
Chiu, Y. H., Hou, S. K., How, C. K., Li, L. H., Kao, W. F., Yang, C. C., et al. (2013). Influence
of a 100-km ultra-marathon on hepatitis B carrier runners. International Journal of Sports
Medicine, 34, 841–845.
Cunniffe, B., Hore, A. J., Whitcombe, D. M., Jones, K. P., Baker, J. S., & Davies, B. (2010).
Time course of changes in immunoendocrine markers following an international rugby game.
Cunniffe, B., Hore, A. J., Whitcombe, D. M., Jones, K. P., Davies, B., & Baker, J. S. (2011).
Immunoendocrine responses over a three week international rugby union series. The Journal
of Sports Medicine and Physical Fitness, 51, 329–338.
Fallon, K. E. (2001). The acute phase response and exercise: The ultramarathon as prototype exer-
cise. Clinical Journal of Sport Medicine, 11, 38–43.
Febbraio, M. A., & Pedersen, B. K. (2002). Muscle-derived interleukin-6: Mechanisms for activa-
tion and possible biological roles. FASEB, 16, 1335–1347.
Frey, W., Wassmer, P., Frey-Rinddova, P., Braun, D., Schwarz, F., Arnold, M., et al. (1994). Muscle
aches and biochemical changes following a ultra-marathon in the cold-modification by diclof-
enac. Schweizerische Zeitschrift für Medizin und Traumatologie, 2, 30–36.
Green, C. L., Brown, L., Stewart, J. J., Xu, Y., Litwin, V., & Mccloskey, T. W. (2011).
Recommendations for the validation of flow cytometric testing during drug development: I
instrumentation. Journal of Immunological Methods, 363, 104–119.
Guissard, N., & Duchateau, J. (2006). Neural aspects of muscle stretching. Exercise and Sport
Sciences Reviews, 34, 154–158.
Kasapis, C., & Thompson, P. D. (2005). The effects of physical activity on serum C-reactive pro-
tein and inflammatory markers. JACC, 45, 1563–1569.
Kim, H. J., Lee, Y. H., & Kim, C. K. (2007). Biomarkers of muscle and cartilage damage and
inflammation during a 200 km run. European Journal of Applied Physiology, 99, 443–447.
Knudson, D. (2006). The biomechanics of stretching. Journal of Exercise Science & Physiotherapy,
2, 3–12.
339–342.
Lund, A. J., Hurst, T. L., Tyrell, R. M., & Thompson, D. (2011). Markers of chronic inflammation
with short-term changes in physical activity. Medicine & Science in Sports & Exercise, 43,
578–583.
Mackiewicz, A., Speroff, T., Ganapathi, M. K., & Kushner, I. (1991). Effects of cytokine com-
binations on acute phase protein production in two human hepatoma cell lines. Journal of
Immunology, 146, 3032–3037.
Marnell, L., Mold, C., & Du Clos, T. W. (2005). C-reactive protein: Ligannds, receptors and role
in inflammation. Clinical Immunology, 117, 104–111.
Mccaffery, M., & Beebe, A. (1989). Pain: Clinical manual for nursing practice. St. Louis, MO:
CV Mosby.
Mcclure, P., Blackburn, L., & Dusold, C. (1994). The use of splints in the treatment of joint stiff-
ness: Biologic rationale and an algorithm for making clinical decisions. Physical Therapy, 74,
1101–1107.
Mcfarland-Mancini, M. M., Funk, H. M., Paluch, A. M., Zhou, M., Giridhar, P. V., Mercer, C. A.,
et al. (2010). Differences in wound healing in mice with deficiency of IL-6 versus IL-6 recep-
tor. Journal of Immunology, 184, 7219–7228.
Moors, M. A., & Mizel, S. B. (2000). Proteasome-mediated regulation of interleukin-1beta turn-
over and export in human monocytes. Journal of Leukocyte Biology, 68, 131–136.
20, 395–406.
Nikolaou, P. K., Macdonald, B. L., Glisson, R. R., Seaber, A. V., & Garrett Jr., W. E. (1987).
Biomechanical and histological evaluation of muscle after controlled strain injury. The
American Journal of Sports Medicine, 15, 9–14.
Noakes, T. D. (1987). Effect of exercise on serum enzyme activities in humans. Sports Medicine,
4, 245–267.
O’Hara, D. M., Xu, Y., Liang, Z., Reddy, M. P., Wu, D. Y., & Litwin, V. (2011). Recommendations
for the validation of flow cytometric testing during drug development: II assays. Journal of
Immunological Methods, 363, 120–134.
Oliver, J. C., Bland, L. A., Oettinger, C. W., Arduino, M. J., Mcallister, S. K., Aquero, S. M., et al.
(1993). Cytokine kinetics in an in vitro whole blood model following an endotoxin challenge.
Lymphokine and Cytokine Research, 12, 115–120.
Paulsen, G., Mikkelsen, U. R., Raastad, T., & Peake, J. M. (2012). Leukocytes, cytokines and
satellite cells: What role do they play in muscle damage and regeneration following eccentric
exercise? Exercise Immunology Review, 18, 42–97.
Pearle, A. D., Scanzello, C. R., George, S., Mandl, L. A., Dicarlo, E., Peterson, M., et al. (2007).
Elevated high-sensitivity C-reactive protein levels are associated with lack inflammatory find-
ings in patients with osteoarthritis. Osteoarthritis and Cartilage, 15, 516–523.
Pepys, M. B., & Hirschfield, G. M. (2003). C-reactive protein: a critical update. The Journal of
Petersen, A. M. W., & Pedersen, B. K. (2005). The anti-inflammatory effect of exercise. Journal of
Pradhan, A. D., Manson, J. E., Rifai, N., Buring, J. E., & Ridker, P. M. (2001). C-reactive protein,
interleukin 6, and risk of developing type 2 diabetus mellitus. JAMA, 286, 327–334.
Prunet, C., Montange, T., Vejux, A., Laubriet, A., Rohmer, J.-F., Riedinger, J.-M., et al. (2006).
Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture
media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients.
Cytometry Part A, 69A, 359–373.
Roberts, W. L., Moulton, L., Law, T. C., Farrow, G., Cooper-Anderson, M., Savory, J., et al. (2001).
Evaluation of nine-automated high sensitivity C-reactive protein methods: Implications for
clinical and epidemiological applications. Part 2. Clinical Chemistry, 47, 418–425.
References 143
Siegel, A. J., Stec, J. J., Lipinska, I., Van Cott, E. M., Lewandrowski, K. B., Ridker, P. M., et al.
(2001). Effect of marathon running on inflammatory and hemostatic markers. The American
Journal of Cardiology, 88, 918–920.
France), 47, 661–673.
Taylor, C., Rogers, G., Goodman, C., Baynes, R. D., Bothwell, T. H., Bezwoda, W. R., et al.
(1987). Hematologic, iron-related, and acute-phase protein responses to sustained strenuous
exercise. Journal of Applied Physiology, 62, 464–469.
Medicine, 18, 300–309.
Weight, L. M., Alexander, D., & Jacobs, P. (1991). Strenuous exercise: Analogous to the acute-
phase response? Clinical Science (London, England), 81, 677–683.
Chapter 4
Study Two: Stretch Intensity vs.
Inflammation: Is There a Dose-Dependent
Association?
Introduction
The parameters of intensity, duration, and frequency feature prominently in stretching

and training programmes, with appropriate combinations believed to produce an
adaptive response (Marschall, 1999; Mujika et al., 1995; Seiler, 2010). With the
magnitude of force applied to a joint during stretching, defined as the intensity of
the stretch, too much force is considered to cause injury to tissue, resulting in
inflammation (Jacobs & Sciacia, 2011). Adopting the view that stretching is a
mechanical force or load, a physical stress, this application of force applied in any
direction (tension, shear, compression) over a given area results in stress to the tis-
sue (stress = force/area) (Mueller & Maluf, 2002).
In response to a protocol of passive stretching, elevated levels of neutrophils
were observed in 3- to 4-month old adult male mice, suggesting an acute inflamma-
tory response (Pizza, Koh, Mcgregor, & Brooks, 2002). Activated neutrophils
secrete pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6) (Gresnigt et al.,
2012), with IL-6, a principal downstream mediator of the acute phase response,
featuring prominently in the release of C-reactive protein (Gabay, 2006; Kilicarslan,
Uysal, & Roach, 2013; Pradhan, Manson, Rifai, Buring, & Ridker, 2001). Since
IL-6 is associated with an inflammatory response, the acute phase changes reflect
the presence and intensity of inflammation, with expression of C-reactive protein
being proportional to the inflammatory stimulus. This response occurs as a feed-
back to trauma, bacterial infection, and collagen tissue disorders (Kilicarslan et al.,
2013). Both IL-6 and C-reactive protein are markers of inflammation (Pradhan
et al., 2001).
Given the importance of stretching on performance, the aim of the present study
was to investigate the effect of various stretching intensities (30, 60, and 90% of
maximum ROM) and their influence on the inflammatory response. We h ypothesise
that low (30% maximum ROM) and medium (60% maximum ROM) compared to

146 4 Study Two: Stretch Intensity vs. Inflammation…
high (90% maximum ROM)-intensity stretching do not elicit and inflammatory

response as measured via the expression of circulating hsCRP (primary outcome).
Methods
Participants
Eleven recreational male athletes (n = 11) were recruited for this study (age,
26 ± 6.20 years.; mass, 85.4 ± 8.55 kg; height, 1.79 ± 0.57 m). Prior to the investiga-
tion, each participant read and signed an informed consent and answered a physical
activity readiness questionnaire (PARQ) and blood analysis questionnaire screening
for any confounding factors (e.g. smoking, injury, chronic disease) that may influ-
ence the outcome. The University of Wolverhampton’s ethics committee granted
ethical approval, with the rights of each participant being protected. On the day
prior to, as well as on the day of the intervention, participants were requested to
refrain from any form of exercise or activity that may cause physical or mental
stress, avoid alcoholic consumption, and ensure that they get adequate sleep.
Power Calculations
The importance of C-reactive protein as an inflammatory biomarker has been previ-

ously assessed pre- and postexercise in stretching (Frey et al., 1994). Assuming a
detectable difference of 2 mg/L with 2 mg/L standard deviation, and an 80% power
with an alpha level of 5%, a sample of 11 participants was required; to allow for
potential drop-outs, we recruited 12 participants (nQuery Advisor v 6.0, Statistical
Solutions, MA, USA).
Procedures
As part of the familiarisation process, during the first visit to the laboratory, the
maximum ROM for the right hamstring muscle was calculated with use of an iso-
kinetic dynamometer (Kin Com, East Ridge, Tennessee, USA). Lying in the supine
position, the right knee of each participant was fitted with a knee immobiliser brace
(Őssur Exoform® knee immobiliser 2220 style, Grjothals, Iceland) (Fig. 4.1),
thereby straightening and locking the knee and preventing knee flexion. With the
pelvis, left leg, and the upper body immobilised, the fulcrum of the lever arm of the
Kin Com was aligned with the greater trochanter of the right leg. The straightened
leg was raised, increasing hip flexion until maximum ROM was achieved, indicated
Methods 147
Fig. 4.1 Knee immobiliser (Published with kind permission of Copyright © Nikos

C. Apostolopoulos 2018. All Rights Reserved)
Fig. 4.2 Experimental set-up (Published with kind permission of Copyright © Nikos

by the sensation of maximal physical pain/effort (Fig. 4.2). The procedure was
repeated three times, with the best value, often the last attempt, recorded as maxi-
mum ROM (Mean = 109.50 ± 20.19). The mean maximum ROM for hip flexion in
males varies with values ranging from 121.3° to 130.4° (Boone & Azen, 1979;
Soucie et al., 2011). Following identification of maximum ROM (independent vari-
able) for each participant, three stretch angle interventions were calculated as 30%,
60%, and 90% of their respective maximum ROM. With use of a computer ran-
domisation programme (www.graphpad.com), all participants were randomised
into each percentage of maximum ROM intervention over 3 consecutive weeks. All
measurements and relevant stretching interventions were conducted in the biome-
chanics laboratory of the University of Wolverhampton.
During the second, third, and fourth visits, participants were informed about
their respective percentage of maximum ROM intervention (i.e. 30%, 60%, or 90%)
for that day (Fig. 4.3). Prior to each intervention, a 5 mL blood sample was collected
from the cephalic vein by a certified phlebotomist. With participants in the supine
position, and their right knee placed and strapped in the immobiliser, their hips,
shoulders, and left lower limb were strapped into the Kin Com preventing internal/
external hip rotation of the right lower limb. To prevent rotation of the right lower
leg in relation to the right femur, the ankle attachment of the Kin Com was used.
© Nikos C. Apostolopoulos 2018. All Rights Reserved)
Methods 149
With the right lower leg resting on the ankle attachment just above the lateral and
medial malleoli, and the foot placed in the neutral position (i.e. no supination or
pronation), the right lower leg was strapped into the ankle rest. To standardise the
set-up and measurement procedures and eliminate bias, designated assistants not
part of the study were assigned to this task for the duration of the study. With the
straightened right lower limb moved into the assigned percentage of maximum
ROM by the dynamometer, the stretch was held for 60 s and then lowered to the start
position for a 10 s rest. The sequence was repeated 5 times in total, with blood col-
lected immediately post and at 24 h post intervention. Since C-reactive protein has
a biological half-life of approximately 19–20 h, falling by up to 50% per day when
the acute stimulus is resolved (Marino & Giotta, 2008; Pepys & Hirschfield, 2003;
Vigushin, Pepys, & Hawkins, 1993), each stretch intervention was conducted on
the same day and hour of the week, ensuring a full week’s rest period. This 1-week
rest period allowed for a full clearance of any potential residual effects, since a sig-
nificant determinant of plasma C-reactive protein levels is the rate of its synthesis,
thereby justifying its use in monitoring the activity of inflammation (Ablij &
Meinders, 2002).
Blood Biomarkers
With blood collected in a vacutainer (BD Vacutainer, K2E EDTA), it rested for
10 min before being spun at 3000 rotations/min in a centrifuge (SciQuip Sigma
2-6E) for 10 min. Since trace amounts of circulating C-reactive protein in healthy
individuals are hardly detectable by standard clinical tests, possessing a detection
limit of 3–8 mg/L (Nanri, Moore, & Kono, 2007; Ridker, 2001), serum hsCRP
(eBioscience—BMS8288FF) was measured by means of a FlowCytomix Simplex
Kit (San Diego, CA, USA) with a Beckman FC500 flow cytometer. This allowed for
a detection level an order of magnitude lower than traditional C-reactive protein
assays (Pearle et al., 2007; Roberts et al., 2001).
Pre-analysis screening using the Kolmogorov-Smirnov test was performed to inves-

tigate the distribution of all variables. Accordingly, LN transformation was used to
overcome skewness and kurtosis in the dependent variable of interest (hsCRP). A
RMANOVA evaluated the changes in hsCRP over time and time × % maximum
ROM, with the LSD post hoc test evaluating any differences based on the observed
main effect. Effect sizes (Cohen’s “d”) were calculated for pre-, post-, and 24 h post
intervention hsCRP for the three stretching interventions comparing 30 vs. 60%
maximum ROM, 30 vs. 90% maximum ROM, and 60 vs. 90% maximum ROM for
our main outcomes of interest (i.e. hsCRP). In addition, a secondary analysis

(linear regression) was conducted investigating the association between percentage
maximum ROM (independent variable) and hsCRP. With level of significance set
at p < 0.05, all statistical analyses were performed via SPSS (version 20.0, SPSS
Inc., USA).
Results
RMANOVA
With all participants involved in the three interventions, no withdrawals or missing

data were recorded. The RMANOVA revealed that Mauchly’s test for sphericity
was violated, for both time (p = 0.001), X2(5) = 13.140, p < 0.05, and for time × %
maximum ROM (p = 0.001), X2(5) = 15.923, p < 0.05. With the degrees of freedom
corrected using the Greenhouse-Geisser estimates of sphericity for time (ε = 0.566)
and time × % maximum ROM (ε = 0.547), significance was revealed for time
(p = 0.011) but not for intensity (p = 0.506). Tests between-subjects effects were
significant (p = 0.001). With time as the observed main effect, an LSD post hoc
analysis reported significant differences between 30 and 90% (p = 0.004) and 60
and 90% (p = 0.034), but not between 30 and 60% (p = 0.089). A comparison of the
means of the LN transformed values for hsCRP for 30% maximum ROM post
(0.475 ± 0.242 mg/L) and for 60% maximum ROM post (0.567 ± 0.237 mg/L) as
well as for 30% maximum ROM 24 h post (0.461 ± 0.232 mg/L) and for 60% maxi-
mum ROM 24 h post (0.578 ± 0.198 mg/L) was quite similar. However, the hsCRP
concentration for 90% maximum ROM post (0.957 ± 0.595 mg/L) and 90% maxi-
mum ROM 24 h post (1.036 ± 0.669 mg/L) increased (Table 4.1).
Table 4.1 Mean (SD) measurement and effect sizes for hsCRP
Mean (SD) for hsCRP (mg/L) Effect sizes (95% CI) between condition comparisons
30% 60% 90% 30 vs. 60% 30 vs. 90% 60 vs. 90%
Pre 0.48 0.57 0.96 −0.39 −1.09 −0.97
(0.25) (0.24) (0.59) (−0.56 to −0.22) (−1.63 to −0.56) (−1.5 to −0.43)
Post 0.48 0.57 0.96 −0.39 −1.11 −0.99
(0.24) (0.24) (0.59) (−0.56 to −0.21) (−1.67 to −0.54) (−1.6 to −0.43)
24 h 0.46 0.58 1.04 −0.46 −0.95 −0.86
post (0.23) (0.19) (0.67) (−0.63 to −0.30) (−1.75 to −0.15) (−1.7 to −0.06)
SD standard deviation
Cohen’s “d” cutoff points for effect size interpretation: small effect ≥0.20, medium effect ≥0.50,
and large effect ≥0.80
Results 151
Effect Sizes
Pre-Intervention hsCRP
Comparison of effect sizes for pre-intervention hsCRP suggests a low effect

(“d” = −0.39; 95% CI, −0.56 to −0.22) when comparing 30 vs. 60% maximum
ROM. However, when comparing 30 vs. 90% as well as 60 vs. 90% maximum
ROM, both comparisons revealed a high effect size (“d” = −1.09; 95% CI, −1.63 to
−0.56 and “d” = −0.97; 95% CI, −1.5 to −0.43, respectively), suggesting that 90%
maximum ROM is associated with a higher inflammatory response (Table 4.1). It is
important to note that the interpretation of these results should be interpreted with
caution, as some discrepancies in the raw data, i.e. baseline hsCRP values in the 30
and 60% conditions, were lower compared to the 90% condition, a fact that may
have influenced the RMANOVA. Please see Table 4.1, which presents these data.
Post Intervention hsCRP
Effect sizes post condition for hsCRP suggest a low effect (“d” = −0.39; 95% CI,
−0.56 to −0.21) when comparing 30 vs. 60% maximum ROM implying that no
major differences exist between these two conditions in terms of the inflammatory
response. However, the comparison between 30 vs. 90% indicates a high effect size
(“d” = −1.11; 95% CI, −1.67 to −0.54), suggesting that as the intensity of the
stretch increases, so does the inflammatory response. Interestingly, a high effect size
(“d” = −0.99; 95% CI, −1.6 to −0.43) was also observed when comparing 60 vs.
90% (Table 4.1).
Post Intervention hsCRP: 24 h
Effect sizes 24 h post intervention hsCRP suggest a low effect size (“d” = −0.46;
95% CI, −0.63 to −0.30) when comparing 30 vs. 60% maximum ROM. However,
when comparing 30 vs. 90% as well as 60 vs. 90% maximum ROM, high effect
sizes were observed (“d” = −0.95; 95% CI, −1.75 to −0.15 and “d” = −0.86; 95%
CI, −1.7 to −0.06, respectively), suggesting a pronounced inflammatory response in
response to stretching intensity (Table 4.1).
econdary Analysis: Linear Regression (30–90 and 60–90%

S
Maximum ROM)
hsCRP: Post
Linear regression analysis was used to test if the % maximum ROM significantly
predicted participants’ hsCRP concentrations. In the analysis of 30% versus 90%
maximum ROM, the results of the regression analysis indicated that % maximum
ROM explained 23.68% of the variance in hsCRP [R2 = 0.24, F(1, 20) = 6.204,
p = 0.022]. Percentage maximum ROM significantly predicted hsCRP [Beta = 0.241,
t(21) = 2.491, p < 0.022]. Therefore, when comparing 30–90% maximum ROM, we
would expect an average increase in hsCRP of 0.24 mg/L (Fig. 4.4).
For the 60% compared to 90% regression analysis, % maximum ROM explained
16.95% of the variance in hsCRP [R2 = 0.17, F(1, 20) = 4.082, p = 0.057]. Percentage
maximum ROM significantly predicted hsCRP [Beta = 0.390, t(21) = 2.021,
p < 0.057]. Therefore, when comparing 60–90% maximum ROM, we would expect
an average increase in hsCRP of 0.39 mg/L (Fig. 4.5).
hsCRP: 24 h Post
For the 30 compared to 90% regression analysis, % maximum ROM explained

26.62% of the variance in hsCRP [R2 = 0.27, F(1, 20) = 7.255, p = 0.014]. Percentage
maximum ROM significantly predicted hsCRP [Beta = 0.288, t(21) = 2.693,
p < 0.014]. Therefore, when comparing 30–90% maximum ROM, we would expect
an average increase in hsCRP of 0.29 mg/L (Fig. 4.6).
The results of the regression analysis comparing 60–90% maximum ROM indi-
cated that % maximum ROM explained 19.12% of the variance in hsCRP [R2 = 0.19,
F(1, 20) = 4.729, p = 0.042]. Percentage maximum ROM significantly predicted
hsCRP [Beta = 0.458, t(21) = 2.175, p < 0.042]. Therefore, when comparing
60–90% maximum ROM, we would expect an average increase in hsCRP of
0.46 mg/L (Fig. 4.7).
2.5
2
hsCRP (mg/L) post
1.5
0.5 R2 = 0.2368
0
20 30 40 50 60 70 80 90 100
% maximum ROM
Unstandardised B = 0.251mg/L
R2 Linear = 0.24
Fig. 4.4 Linear regression between 30 and 90% maximum ROM and hsCRP post
Results 153
2.5
2
hsCRP (mg/L) post
1.5
0.5
R2 = 0.1695
0
50 55 60 65 70 75 80 85 90 95
% maximum ROM
R2 Linear = 0.17
Fig. 4.5 Linear regression between 60 and 90% maximum ROM and hsCRP post
2.5
hsCRP (mg/L) 24hrs post
1.5
0.5 R2 = 0.2662
0
20 30 40 50 60 70 80 90 100
% maximum ROM
R2 Linear = 0.27
Fig. 4.6 Linear regression between 30 and 90% maximum ROM and hsCRP 24 h post
2.5
hsCRP (mg/L) 24hrs post
1.5
0.5 R2 = 0.1912
0
50 55 60 65 70 75 80 85 90 95
% maximum ROM
R2 Linear = 0.19
Fig. 4.7 Linear regression between 60 and 90% maximum ROM and hsCRP 24 h post
Discussion
The effects of different percentages of maximum ROM on hsCRP, a biomarker for

inflammation, were investigated. Results suggest that stretching between 30 and
60% maximum ROM did not elicit an increase in blood hsCRP concentrations, with
a marked increase in hsCRP observed contrasting 30–90% and 60–90% maximum
ROM immediately post and 24 h post stretch interventions. Effect sizes revealed a
pronounced inflammatory response comparing both 30 and 60–90% maximum
ROM. In addition, the secondary analysis suggests a moderate positive relationship
for both hsCRP post and 24 h post.
The extent of muscle tissue disruption is dependent on the intensity and the
severity of the exercise (Tiidus & Ianuzzo, 1983), with stretching injuries associated
with an initial mechanical perturbation, followed by a secondary or collateral injury,
coincident with neutrophil invasion and the synthesis of IL-6 (Toumi, F’guyer, &
Best, 2006). The ubiquitous IL-6, associated with the control and co-ordination of
immune response, is primarily responsible for activation of the acute phase response
with the synthesis of C-reactive protein (Gabay, 2006; Kilicarslan et al., 2013;
Toumi et al., 2006). Marked increases in the concentration of hsCRP post and 24 h
post, comparing 30 and 60–90% maximum ROM, suggest that intense stretching
was responsible for inflammation in lieu of a mechanical stress on muscle tissue.
However, since baseline values for both 30 and 60% compared to 90% for hsCRP
were considerably different, one needs to exercise caution (Table 4.1). This observed
Conclusion 155
anomaly may be a result of mental stress prior to the intense stretching intervention,
alcohol consumption, infection, or lack of sleep prior to assessment. Unfortunately,
we were not able to identify which one of these reasons resulted in this discrepancy,
as all participants reported that they indeed followed the recommendations provided
by the principal researcher the day prior to the assessment while they were all appar-
ently healthy (infection-free).
Stretching intensity is difficult to measure quantitatively, as opposed to duration
(amount of time) and frequency (how often), accounting for the dearth of studies
(Apostolopoulos, Metsios, Flouris, Koutedakis, & Wyon, 2015). Assessment of the
degree of intensity of a stimulus is highly variable (Melzack & Katz, 1999) defined
by the power law proposed by Stevens (Baliki, Geha, & Apkarian, 2009). According
to Stevens, sensation magnitudes grow as power functions of stimulus intensities
producing them, describing relationships between human sensations, subjective
impressions, and the physical stimuli evoking them (Zwislocki, 2009). This func-
tional relation between the subjective and physical magnitude of a mechanical force
(Stevens & Mack, 1959) is an internal, outwardly directed, “adaptive,” or “match-
ing” response generated within the organising system (MacKay, 1963). Regardless
of the difficulty in measuring stretching intensity, its role on maximum jump height
was investigated, with an intensity level of <50% of point of discomfort, prior to
performance, observed to be beneficial (Behm & Kibele, 2007). Stretching to a
maximal point of discomfort was responsible for muscle damage, similar to effects
seen with eccentric-induced damage associated with DOMS (Behm & Kibele,
2007). In agreement with the aforementioned study, stretching intensities associated
with 30 and 60 did not exhibit an inflammatory response (i.e. rise in hsCRP) com-
pared to 90% of maximum ROM. However, to investigate this relationship further,
future research needs to measure plasma creatine kinase (CPK), a marker of muscle
damage, with increases related to both the intensity and duration of exercise
(Noakes, 1987).
The secondary analysis revealed a moderate positive correlation between the
independent variables (30, 60, and 90% maximum ROM) and the dependent vari-
able (hsCRP). The increase in the concentration of hsCRP associated with the dif-
ferent percentages of maximum ROM potentially suggests a method for quantifying
stretch intensity using a blood biomarker. More research is needed to verify such a
hypothesis.
Conclusion
The current data revealed that increases in percentage maximum ROM were associ-
ated with a progressive increase in hsCRP. Since the production of hsCRP has been
linked to inflammation, intensities between 30 and 60% maximum ROM were less
likely to cause inflammation. However, although these results were confirmed by
our collective analyses (RMANOVA and effect sizes), these results should be treated
with caution, due to some unexpected discrepancies in the hsCRP baseline values
between the three conditions, suggesting further research.
References
Ablij, H. C., & Meinders, A. E. (2002). C-reactive protein: History and revival. European Journal
of Internal Medicine, 13, 412–422.
Baliki, M. N., Geha, P. Y., & Apkarian, A. V. (2009). Parsing pain perception between nociceptive
representation and magnitude estimation. Journal of Neurophysiology, 101, 875–887.
Boone, D. C., & Azen, S. P. (1979). Normal range of motion of joints in male subjects. Journal of
Bone and Joint Surgery (American Volume), 61, 756–759.
Frey, W., Wassmer, P., Frey-Rinddova, P., Braun, D., Schwarz, F., Arnold, M., et al. (1994). Muscle
aches and biochemical changes following a ultra-marathon in the cold-modification by diclof-
enac. Schweizerische Zeitschrift für Medizin und Traumatologie, 2, 30–36.
Gresnigt, M. S., Joosten, L. A. B., Verschueren, I., Van Der Meer, J. W. M., Netea, M. G., Dinaello,
C. A., et al. (2012). Neutrophil inhibition of proinflammatory cytokine responses. Journal of
Immunology, 189, 4806–4815.
Mackay, D. M. (1963). Psychophysics perceived intensity: A theoretical basis for Fechner's and
Steven's laws. Science, 139, 1213–1216.
Melzack, R., & Katz, J. (Eds.). (1999). Pain measurements in persons with pain. London, UK:
Chruchill Livingstone.
Mueller, M. J., & Maluf, K. (2002). Tissue adaptation to physical stress: A proposed “physical
stress theory” to quide physical therapist practice, education, and research. Physical Therapy,
82, 383–403.
20, 395–406.
Nanri, A., Moore, M. A., & Kono, S. (2007). Impact of C-reactive protein on disease risk and its
relation to dietary factors: Literature review. Asian Pacific Journal of Cancer Prevention, 8,
167–177.
4, 245–267.
Pearle, A. D., Scanzello, C. R., George, S., Mandl, L. A., Dicarlo, E., Peterson, M., et al. (2007).
Elevated high-sensitivity C-reactive protein levels are associated with lack inflammatory find-
ings in patients with osteoarthritis. Osteoarthritis and Cartilage, 15, 516–523.
Pepys, M. B., & Hirschfield, G. M. (2003). C-reactive protein: A critical update. The Journal of
Physiology, 92, 1873–1878.
Pradhan, A. D., Manson, J. E., Rifai, N., Buring, J. E., & Ridker, P. M. (2001). C-reactive protein,
interleukin 6, and risk of developing type 2 diabetus mellitus. JAMA, 286, 327–334.
References 157
Ridker, P. M. (2001). High-sensitivity C-reactive protein, potential adjunct for global risk
assessment in the primary prevention of cardiovascular disease. Circulation, 103, 1813–1818.
Roberts, W. L., Moulton, L., Law, T. C., Farrow, G., Cooper-Anderson, M., Savory, J., et al. (2001).
Evaluation of nine-automated high sensitivity C-reactive protein methods: Implications for
clinical and epidemiological applications. Part 2. Clinical Chemistry, 47, 418–425.
Seiler, S. (2010). What is best practice for training intensity and duration distribution in endurance
athletes? International Journal of Sports Physiology and Performance, 5, 276–291.
Soucie, J. M., Wang, C., Forsyth, A., Funk, S., Denny, M., Roach, K. E., et al. (2011). Range of
motion measurements: Reference values and a database for comparison studies. Haemophilia,
17, 500–507.
Stevens, J. C., & Mack, J. D. (1959). Scales of apparent force. Journal of Experimental Psychology,
58, 405–413.
Tiidus, P., & Ianuzzo, C. (1983). Effects of intensity and duration of muscular exercise on delayed
onset muscle soreness and serum enzymes activities. Medicine and Science in Sports and
Exercise, 15, 461–465.
Toumi, H., F’guyer, S., & Best, T. M. (2006). The role of neutrophils in injury and repair following
muscle stretch. Journal of Anatomy, 208, 459–470.
Zwislocki, J. J. (2009). Sensory neuroscience: Four laws of psychophysics. New York: Springer.
Chapter 5
The Effect of Different Passive Static
Stretching Intensities on Perceived Muscle
Soreness and Muscle Function Recovery
Following Unaccustomed Eccentric
Exercise: A Randomised Controlled Trial
Introduction
The first study proposed that high-intensity passive static stretching (IS) was
affiliated with a pronounced inflammatory response, with study 2 suggesting that
stretching between 30 and 60% of an individual’s maximum ROM does not elicit
inflammation and an inflammatory response. Given that the practical application of
stretching includes the amelioration of some post-training effects, such as muscle
soreness, decrease in muscle strength, and inflammation, the focus of the third study
was to investigate the effects of low- versus high-intensity passive static stretching
and no stretching, on recovery from unaccustomed eccentric exercise.1
DOMS refers to the sensation of muscular discomfort and pain in response to
eccentric muscle contractions, as well as exhaustive and unaccustomed, or high-
intensity exercise (Kanda et al., 2013; Paschalis, Nikolaidis, et al., 2007). Intensity
of discomfort associated with DOMS increases within the first 24 h, peaks between
24 and 72 h, eventually disappearing 5–7 days postexercise (Armstrong, 1984;
Talag, 1973). The perception of muscle soreness associated with DOMS is to be due
to the activation of free nerve endings around muscle fibres serving as receptors of
noxious stimuli associated with muscle damage (Byrnes & Clarkson, 1986). In
addition to soreness, structural damage to muscle caused by eccentric exercise alters
its function and joint mechanics (Rowlands, Eston, & Tilzey, 2001).
Soreness associated with DOMS has negative consequences temporarily restrict-
ing the ability to perform an activity (Vasudevan, 1993). DOMS has been associ-
ated—inter alia—with a loss of maximum voluntary force (Clarkson, Nosaka, &
Braun, 1992; Newham, Jones, & Clarkson, 1987; Newham, Mills, Quigley, &
Edwards, 1983), changes in the contractile properties of muscle (Newham et al.,
1983), increased protein levels related to muscle damage (i.e. creatine kinase)
(Clarkson et al., 1992; Newham et al., 1987; Nosaka & Clarkson, 1992; Peake,
Parts of this chapter appear in Apostolopoulos et al., (2018).

1

160 5 The Effect of Different Passive Static Stretching Intensities on Perceived Muscle…
Nosaka, & Suzuki, 2005), and decreased running economy (Paschalis, Koutedakis,
Jamurtas, Mougios, & Baltzopoulos, 2005). Disruption is influenced by three fac-
tors: the magnitude of the response, the previous history of muscle use, and the
injury-specific interactions between muscle and the invading inflammatory cells
(Tidball, 2005), suggesting that the ability to alleviate this process partly resides in
the act of diminishing the magnitude of inflammation and the inflammatory
response. Since soreness associated with DOMS affects athletic performance, a
strategy introduced to diminish its severity sooner, aids in restoration of the maxi-
mal function of muscle (Cheung, Hume, & Maxwell, 2003). Early alleviation from
the symptoms associated with DOMS postexercise may also increase adherence to
regular exercise by nonathletes (Smith et al., 1993).
Muscular actions, such as unaccustomed eccentric muscle exercise, of sufficient
intensity and duration, have been repeatedly verified in human and animal studies as
being responsible for the disruption of contractile and connective tissue (Friden,
Sjostrom, & Ekblom, 1983; Lieber & Friden, 1993; Smith et al., 1993). DOMS
activated in response to tissue injury is a form of acute inflammation with the sensa-
tion of soreness representing inflammatory pain (Lieber & Friden, 1993; Macintyre,
Reid, & Mckenzie, 1995; Smith, 1991). Disruption of muscle tissue by unaccus-
tomed eccentric exercise is characterised by an initial mechanical and a secondary
biochemical injury, with the former associated with a disorganisation of the myofi-
brillar material, in particular, a focal disruption of the sarcomeres (Armstrong,
1984; Faulkner, Brooks, & Opiteck, 1993), with the Z-disc being the most vulner-
able structure (Friden & Lieber, 2001). The biochemical injury, characterised by a
“local reaction” at the site of damage, results in accumulation of neutrophils and
macrophages, with the magnitude related to both the intensity and duration of the
exercise (Castell, Andus, Kunz, & Heinrich, 1989; Peake, 2002; Peake et al., 2005;
Robson et al., 1999; Tiidus & Ianuzzo, 1983). Neutrophils appear within hours post-
unaccustomed eccentric exercise, remaining for up to 48 h (Beaton, Tarnopolsky, &
Phillips, 2002; Frenette, Cai, & Tidball, 2000; Malm et al., 2000; Macintyre, Reid,
Lyster, & Mckenzie, 2000; Stupka, Tarnopolsky, Yardley, & Phillips, 2001), with
macrophages appearing at 24 h and remaining present for up to 14 days (Beaton,
Allan, Tarnopolsky, Tiidus, & Phillips, et al., 2002; Beaton, Tarnopolsky et al.,
2002; Malm et al., 2000; Peake et al., 2005). These inflammatory cells release the
pro-inflammatory cytokines, IL-1β, TNF-α, and IL-6 (Castell et al., 1989). As men-
tioned in sections above, these cytokines are part of a network with the expression
of each being influenced by the other (Shizuo, Hirano, Taga, & Kishimoto, 1990),
with IL-6 being the primary mediator for the increased expression of C-reactive
protein (Bauer, Lengyel, Bauer, Acs, & Gerok, 1989; Castell et al., 1988; Castell
et al., 1989; Kushner & Rzewnicki, 1994).
Attempts have been made to identify treatments for DOMS, including use of
non-steroidal anti-inflammatory drugs (Hasson et al., 1993; Hertel, 1997; Janssen,
Kuipers, Vertsappen, & Costill, 1983), massage (Lightfoot, Char, Mcdermott, &
Goya, 1997; Weber, Servedio, & Woodall, 1994), cryotherapy (Gullick & Kimura,
1996), physical activity (Hasson, Barnes, Hunter, & Williams, 1989), and static
Methods 161
stretching (High, Howley, & Franks, 1989; Johansson, Lindstrom, Sundelin, &
Lindstrom, 1999; Lund, Vestergaard-Poulsen, Kanstrup, & Sejrsen, 1998; Maxwell,
Kohl, Watson, & Belnave, 1988; Wessel & Wan, 1994), with investigations on
stretching as a treatment modality for DOMS focused on whether stretching can
prevent or alleviate DOMS (High et al., 1989, Johansson et al., 1999, Lund et al.,
1998, Wessel & Wan, 1994). Studies investigating the effects of pre- and postexer-
cise static stretching on DOMS found no preventive effect on muscular soreness,
tenderness, and force loss from unaccustomed eccentric exercise (High et al., 1989;
Johansson et al., 1999). Similarly, a systematic review, based on eligible randomised
controlled trials, concluded that regardless of when stretching was performed, no
clinically important reductions in DOMS occurred (Herbert, De Noronha, &
Kamper, 2011). However, none of these studies considered the effect of stretching
intensity.
Therefore, the objective of this study was to investigate the effect of low- and
high-intensity passive static stretching compared to no stretching (control). The pri-
mary aim investigated effects on perceived muscle soreness associated with unac-
customed eccentric exercise of the right knee extensors, with the secondary aim
examining effects on muscle function (eccentric and isometric peak torque). We
hypothesised that low-intensity passive static stretching, rendered over 3 consecu-
tive days, would lower perceived muscle soreness and improve muscle function
compared to high-intensity passive static stretching and no stretching.
Methods
Participants
Thirty recreationally active male participants (age, 25 ± 6 years; mass,

83.1 ± 10.7 kg; height, 1.78 ± 0.68 m) were recruited and assessed for 4 consecu-
tive days: at baseline, 24, 48, and 72 h. All participants were actively involved in
resistance training on a regular basis and were familiar with the concept of per-
forming maximal contractions, with institutional ethical approval granted by the
University of Wolverhampton. Each participant read and signed the informed con-
sent form, completing a physical activity readiness questionnaire (PARQ) and a
blood analysis screening form prior to investigation, looking for factors that may
influence the investigation (e.g. alcohol, coffee, medication, smoking, activity lev-
els). The rights of each participant were protected. Additional exclusion criteria
included any form of physical limitations regarding hips, knees, and ankles.
Following the unaccustomed eccentric exercise, participants were advised to
refrain from any form of physical activity, not consume alcohol, coffee, medica-
tion, abstain from smoking, and to ensure they get a good night’s sleep for the
duration of the study.
© Nikos C. (Apostolopoulos, 2018). All Rights Reserved)
Procedures
The 30 participants were randomly allocated, using an online randomisation

programme (www.graphpad.com), into one of three groups: (1) low-intensity pas-
sive static stretching (30–40% of maximum perceived stretch), (2) high-intensity
passive static stretching (70–80% of the individual’s maximum perceived stretch),
or (3) no stretching (control), consisting of ten participants per group. There were
no withdrawals from this investigation.
The working definition of a “maximum perceived stretch” refers to a perception
associated with pain and discomfort during passive static stretching, measured on a
numerical rating scale anchored at 0 (i.e. no pain) and ranging to 10 (i.e. extreme
pain), with “maximum perceived stretching” being equivalent to a value of 10. On
arrival to the laboratory, participants’ anthropometric measurements [mass (kg) and
height (m)] were taken. Figure 5.1 highlights the experimental process.
Methods 163
Pre-unaccustomed Eccentric Exercise Assessment
Participants were assessed at baseline (time 0) and at 24, 48, and 72 h pre-
unaccustomed eccentric exercise, for eccentric and isometric peak torque of the
right knee extensors on a KinCom isokinetic dynamometer (KinCom, www.kin-
com.com, East Ridge, Tennessee, USA). Muscle soreness/DOMS was assessed
immediately following unaccustomed eccentric exercise and again at 24, 48, and
72 h postexercise, prior to eccentric and isometric peak torque tests.
Alignment of the rotational axis of the dynamometer and the right knee joint
rotational axis (lateral femoral epicondyle) was set for each participant, with the
ankle cuff being attached proximal to the lateral malleolus. In addition, each partici-
pant’s upper body and left quadriceps were securely fastened before assessment.
Familiarisation consisted of five submaximal repetitions for each condition, with
participants requested to apply 50% of maximal effort, immediately followed by a
1-min rest prior to the maximal effort for both eccentric and isometric peak torque.
Eccentric peak torque was measured at 1.05 rad/s (i.e. 60° per second), with the
lower leg moved through a range of motion starting at 20°, progressing to 100° of
knee flexion. Isometric peak torque of the right knee flexion muscles was measured
with the knee held at 60° of knee flexion. To ensure standardisation for the proce-
dure, force output was closely monitored on the KinCom display by an assistant.
During three maximal efforts, verbal encouragement was provided by an assistant
not part of the study to eliminate bias, with the highest value being recorded (often
the third attempt).
Eccentric Exercise Protocol
Following a 5-min rest period, after the assessment for both eccentric and isometric
peak torque, participants were familiarised with the eccentric exercise protocol for
the right knee extensors. This consisted of six sets of ten eccentric repetitions
through a range of motion from 20 to 100° of knee flexion at a speed of 1.05 rad/s,
with a 2-min rest between sets, as previously described (Paschalis, Giakas, et al.,
2007). The familiarisation process consisted of a single set of ten submaximal rep-
etitions using an identical setup as that for the unaccustomed eccentric exercise
protocol. After a 1-min rest, participants began the eccentric exercise protocol. To
ensure standardisation for the procedure for each group, the force output was closely
monitored on the KinCom display by an assistant, whom provided verbal encour-
agement, and was not part of the study to minimise bias.
Upon completion, participants were asked to rate their perceived muscle sore-
ness on a numerical rating scale anchored at 0 (i.e. no soreness) to 10 (i.e. extremely
sore), by palpating the distal region of the relaxed right knee extensors while seated.
They returned to the laboratory at 24, 48, and 72 h following the unaccustomed
eccentric exercise for both eccentric and isometric peak torque tests, and the record-
ing of their perceived muscle soreness levels prior to the muscle function tests for
both eccentric and isometric peak torque.
Passive Static Stretching Exercise Protocol
Following the unaccustomed eccentric exercise protocol, participants in both the

low- and high-intensity stretching groups performed an identical passive static
stretching protocol bilaterally, for the hamstrings, hip flexors, and quadriceps mus-
cle groups (in that order) for 3 consecutive nights prior to sleep (Figs. 5.2, 5.3, and
5.4). Although only the knee extensor muscle group was exposed to the unaccus-
tomed eccentric exercise, this group has an antagonistic relationship to the ham-
string muscle group, with both influenced by the amount of hip flexion (Worrel,
Perrin, & Denegar, 1989).
During the participants’ first day at the laboratory, a trained therapist provided
one-on-one instruction on the proper form and execution of the three passive static
stretching exercises, to be performed at home before sleep for three consecutive days.
Unlike the previous two studies, where participants and the principle investigator had
Fig. 5.2 Passive static stretching exercises (published with kind permission of copyright © Nikos
Methods 165
direct contact, and monitoring was possible for maintenance of the correct intensity
of the stretch, participants performed the stretches at home. Therefore, a range of
stretching threshold (30–40% and 70–80% of maximum perceived stretch) was
introduced to provide participants with the opportunity to interpret the stretch sensa-
tion; this approach closely simulates clinical and sport performance approaches (the
patient or the athlete stretches to a volitional range of motion). By presenting both a
range and a description of the sensation of the stretch (i.e. gentle, warm, pain, dis-
comfort, etc.), this conveyed the magnitude of the stretch, a form of a “magnitude
production” (Stevens, 1971), allowing participants to perform the given task
(stretches between 30–40% and 70–80% of maximum perceived stretch) based on
their interpretation.
Using a numerical rating scale (anchored at 0—no pain to 10—extreme pain),
both stretching groups performed a maximum stretch to the point of extreme pain.
Based on this perceived sensation and with use of the numerical rating scale (i.e.
magnitude production), the low-intensity stretching group was taught to maintain an
intensity level of 3–4 out of 10 (i.e. a warm gentle feeling), with the high-intensity
stretching group maintaining an intensity level of a 7–8 out of 10 (i.e. discomfort/
slight pain). To reinforce the instructions given, a handout on how to perform the
exercises was provided (see Appendix). In addition, during the subsequent visits to
the laboratory for the eccentric and isometric peak torque tests (24 and 48 h), par-
ticipants were questioned about their respective stretching protocol by the therapist,
particularly if they maintained the required stretching intensity level assigned. Each
stretch was supported with use of a bench or pillow, allowing for better isolation and
targeting of the muscle group being stretched. Support of the muscle group mini-
mises muscular contraction from occurring during the stretch (Abdel-Aziem, Draz,
Mosaad, & Abdelraou, 2013; Apostolopoulos, 2010). With the duration of each
passive static stretch being 60 s, and repeated three times per muscle group per side
(i.e. a total of 6 min per stretch exercise), the complete stretching routine lasted
approximately 18 min.
Routine pre-analyses screening using the Shapiro-Wilks test was performed to

investigate the distribution of all the variables. A two-way ANOVA with repeated
measures for time (0, 24, 48, and 72 h) and condition (low- and high-intensity
stretching and control) was performed on the variables for perceived muscle sore-
ness and muscle function (eccentric and isometric peak torque). Post hoc t-tests
with Bonferroni adjustment for multiple comparisons were performed when sig-
nificant main effects of time were detected. The level of statistical significance
was set at p < 0.05. All statistical analyses were performed via SPSS software
(version 20.0).
To detect small effects of practical performance, data for the three conditions
was analysed using a contemporary magnitude-based inference approach (Hopkins,
2007). Each outcome was analysed via a spreadsheet (Hopkins, 2006) that used log
transformation to estimate the effect of the conditions (low- vs. high-intensity pas-
sive static stretching and both low- and high-intensity passive static stretching vs.
control), across each 24-h period [before, or immediately after, unaccustomed
eccentric exercise (for perceived muscle soreness) to 24, 24 to 48, and 48 to 72 h] as
the difference in the mean percentage change, as well as in Cohen d units. The
thresholds for small, moderate, large, and very large effects (Cohen d) were 0.2, 0.6,
1.2, and 2.0 (Batterham & Hopkins, 2005). To estimate the chances that the stretch-
ing interventions had a beneficial, trivial, or harmful effect, a Cohen unit of 0.2 was
employed as the smallest meaningful effect on the dependent variables. Where the
chance of benefit and harm are both >5%, the effect was deemed unclear. Qualitative
descriptors were then assigned to quantitative percentile scores as follows: 25–75%
possible, 75–95% likely, and > 99% most likely (Batterham & Hopkins, 2005;
Hopkins, 2002, 2006). For comparisons that were unclear, we reported the effect
(i.e. beneficial, trivial, or harmful) with the highest percentage. For each analysis,
the effect was adjusted for the outcome’s baseline grand mean.
Results
Perceived Muscle Soreness

RMANOVA
Although no statistical difference was detected for a time x condition interaction

(p = 0.573), a statistically significant main effect of time on perceived muscle sore-
ness values after an unaccustomed eccentric exercise bout was observed (p < 0.001),
suggesting a reduction in perceived muscle soreness values over time regardless of
condition (Table 5.1, Fig. 5.3). In addition, distribution of perceived muscle sore-
ness as a function of time revealed that low-intensity passive static stretching was
associated with a faster decrease in soreness levels (Fig. 5.4).
Magnitude-Based Inference Approach
Low- vs. High-Intensity Passive Static Stretching
The results of the magnitude-based inference analysis are presented in Tables 5.2,
5.3, and 5.4. Assuming that a small effect is meaningful (i.e. Cohen’s d = 0.2), low-
intensity passive static stretching resulted in a likely small beneficial reduction in
perceived muscle soreness immediately following unaccustomed eccentric exercise
(time 0) to 24 h, an unclear effect between 24 and 48 h and a likely moderate
beneficial reduction in muscle soreness compared with high-intensity passive static
stretching between 48 and 72 h. However, low- vs. high-intensity passive static
Results
Table 5.1 Measurements of perceived muscle soreness, eccentric and isometric peak torque over 72 h post-unaccustomed eccentric exercise protocol (values are
mean ± SD; n = 10 per group)
Soreness Eccentric peak torque (Nm) Isometric peak torque (Nm)
Condition 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h 0 h 24 h 48 h 72 h
Low- 6.0 ± 1.4 5.0 ± 1.3 2.9 ± 1.2 1.2 ± 0.4 247.5 ± 62.0 229.6 ± 62.8 244.3 ± 53.1 263.1 ± 61.9 207.6 ± 40.2 196.4 ± 46.2 209.5 ± 47.0 222.3 ± 47.9
intensity
passive
static
stretching
(LIS)
High- 5.9 ± 1.8 5.7 ± 2.2 3.7 ± 1.4 2.4 ± 1.3 218.2 ± 59.7 173.4 ± 35.6 208.0 ± 44.7 195.9 ± 31.9 181.3 ± 41.2 163.5 ± 41.7 172.7 ± 50.1 186.6 ± 39.1
intensity
passive
static
stretching
(HIS)
Control 5.9 ± 1.8 5.7 ± 1.9 4.0 ± 1.3 2.2 ± 1.3 214.8 ± 52.7 196.2 ± 49.8 179.4 ± 42.8 200.6 ± 65.6 185.1 ± 55.2 165.1 ± 49.5 169.6 ± 50.6 172.8 ± 55.4
SD standard deviation
167
Fig. 5.3 Perceived muscle

soreness (raw)
stretching most likely had a large beneficial effect on perceived muscle soreness
when time 0 was compared with 72 h (see Table 5.2).
Low-Intensity Passive Static Stretching vs. Control
Low-intensity passive static stretching resulted in likely small and very likely large
beneficial decrease in perceived muscle soreness from immediately post-
unaccustomed eccentric exercise to 24 and 72 h after, respectively. The effects of
low-intensity passive static stretching compared with control at subsequent time
points were unclear (see Table 5.3).
High-Intensity Passive Static Stretching vs. Control
The effect of high-intensity passive static stretching versus control on perceived

muscle soreness was unclear between all assessed time points (see Table 5.4).
Eccentric Peak Torque

RMANOVA
A statistically significant main effect for time on eccentric peak torque (p < 0.001)
was observed, indicating that scores differ across time points. The time x condition
interaction effect was significant (p = 0.008), indicating that eccentric peak torque
scores differed across assessment time points for the three conditions. Simple main
Results 169
Fig. 5.4 Distribution of

perceived muscle soreness
as a function of time
Table 5.2 Changes in perceived muscle soreness, eccentric and isometric peak torque in the
low-intensity passive static stretching (LIS) vs. high-intensity passive static stretching (HIS) group
(n = 10 per group)
LIS vs. HIS LIS vs.
LIS % % change, HIS
change, HIS % change, MD [90% effects, Qualitative
Outcomes Time MD ± SD MD ± SD CI] [90% CI] inference
Perceived 0–24 ha −18.3 ± 30.1 −6.7 ± 37.7 −12.4 −0.27 Possibly
muscle [−30.4, [−0.74, beneficial
soreness 10.2] 0.20] (60%)
24– −44.3 ± 63.6 −32.8 ± 60.3 −17.2 −0.39 Unclear
48 h [−43.2, [−1.15,
20.8] 0.39]
48– −56.7 ± 73.0 −37.1 ± 46.4 −31.1 −0.76 Likely
72 h [−52.5, [−1.52, beneficial
−0.1] 0.00] (89%)
0–72 h −80.3 ± 35.6 −60.6 ± 60.7 −50.0 −1.41 Most likely
[−63.6, [−2.06, beneficial
−31.5] −0.77] (100%)
Eccentric Pre- −7.1 ± 12.4 −23.5 ± 20.3 21.5 [7.0, 0.71 Very likely
peak torque 24 h 37.8] [0.25, beneficial
(Nm) 1.16] (96%)
Pre- 1.4 ± 12.4 −6.1 ± 17.7 8.0 [−3.7, 0.28 Possibly
48 h 21.2] [−0.14, beneficial
0.70] (63%)
Pre- 7.7 ± 10.0 −12.8 ± 15.4 23.4 [11.8, 0.76 Very likely
72 h 36.3] [0.40, beneficial
1.12] (99%)
Isometric Pre- −7.1 ± 8.0 −10.4 ± 13.4 3.6 [−5.0, 0.14 Possibly
peak torque 24 h 13.0] [−0.20, trivial (57%)
(Nm) 0.48]
Pre- 0.4 ± 13.6 −5.2 ± 15.1 5.9 [−5.2, 0.23 Possibly
48 h 18.3] [−0.21, beneficial
0.66] (54%)
Pre- 7.0 ± 10.4 −17.5 ± 145.1 29.8 1.03 Unclear
72 h [−25.8, [−1.18,
127.2] 3.24]
MD mean difference, SD standard deviation (presented as a CV %), 90% CI 90% confidence inter-
val, d Cohen’s d effect size
a
0–24 h = pre-unaccustomed eccentric exercise to 24 h post-unaccustomed eccentric exercise
effects analysis revealed that all three conditions statistically changed over time
(high-intensity, p = 0.001; low-intensity, p = 0.024; control, p = 0.014), whereas
statistical significant between-group differences were observed at 48 (p = 0.0170
and 72 h (p = 0.019), but not at baseline (p = 0.399) or 24 h (p = 0.061) (Table 5.1,
Fig. 5.5). The histogram below (Fig. 5.6) revealed changes between the three condi-
tions with respect to baseline. Eccentric peak torque decreased from baseline to
Results 171
low-intensity passive static stretching (LIS) vs. control (CON) group (n = 10 per group)
LIS vs.
CON % LIS vs.
LIS % CON % change, CON
change, change, MD [90% effects, d Qualitative
Outcomes Time MD ± SD MD ± SD CI] [90% CI] inference
Perceived 0–24 ha −18.3 ± 30.1 −4.5 ± 32.7 −14.4 −0.46 Likely
muscle [−30.9, 6.0] [−1.10, beneficial
soreness 0.17] (76%)
24– −44.0 ± 63.6 −28.9 ± 37.4 −21.3 −0.71 Unclear
48 h [−43.3, 9.3] [−1.69,
0.26]
48– −56.7 ± 73.0 −51.1 ± 74.9 −11.5 −0.36 Unclear
72 h [−42.7, [−1.65,
36.6] 0.93]
0–72 h −80.2 ± 35.6 −66.8 ± 88.4 −40.4 −1.54 Very likely
[−60.0, [−2.73, beneficial
−11.1] −0.35] (97%)
Eccentric Pre- −7.0 ± 12.4 −9.1 ± 12.4 2.3 [−7.0, 0.09 Unclear
peak torque 24 h 12.6] [−0.27,
(Nm) 0.44]
Pre- 1.6 ± 12.4 −17.4 ± 17.6 23.0 [9.5, 0.77 [0.34, Very likely
48 h 38.2] 1.20] beneficial
(98%)
Pre- 7.8 ± 10.0 −6.9 ± 15.6 15.8 [4.6, 0.54 [0.17, Likely
(93%)
Isometric Pre- −7.1 ± 8.0 −11.8 ± 20.7 5.5 [−6.4, 0.19 Unclear
peak torque 24 h 18.9] [−0.24,
(Nm) 0.62]
Pre- 0.3 ± 13.6 −9.8 ± 24.1 11.2 [−3.7, 0.38 Possibly
48 h 28.4] [−0.14, beneficial
0.91] (73%)
Pre- 7.1 ± 10.4 −8.4 ± 26.1 16.8 [0.7, 0.56 [0.03, Likely
(88%)
Analyses were adjusted to baseline grand means for each outcome
a
24 h post-unaccustomed eccentric exercise in all groups, with only the low-intensity
passive static stretching group increasing consistently from 24 to 72 h. In addition,
the scatter plot (Fig. 5.7) suggests that low-intensity passive static stretching is asso-
ciated with an increase in eccentric peak torque (72–0 h) compared to the other two
conditions.
high-intensity passive static stretching (HIS) vs. control (CON) group (n = 10 per group)
HIS vs.
CON % HIS vs.
CON % change, CON
HIS % change, change, MD [90% effects, d Qualitative
Outcome Time MD ± SD MD ± SD CI] [90% CI] inference
Perceived 0–24 ha −6.6 ± 37.7 −4.4 ± 32.7 −2.3 −0.04 Unclear
muscle [−22.9, [−0.51,
soreness 23.9] 0.42]
24– −32.6 ± 60.3 −28.9 ± 37.4 −5.3 −0.11 Unclear
48 h [−31.0, [−0.73,
30.0] 0.51]
48– −36.9 ± 46.4 −51.1 ± 74.9 29.0 0.50 Unclear
72 h [−11.5, [−0.24,
88.1] 1.24]
0–72 h −60.3 ± 60.7 −66.7 ± 88.4 19.4 0.35 Unclear
[−23.2, [−0.52,
85.6] 1.21]
Eccentric Pre- −18.8 ± 20.3 −9.1 ± 12.4 −10.6 −0.46 Likely
peak torque 24 h [−20.9, 1.1] [−0.97, harmful
(Nm) 0.04] (81%)
Pre- −3.5 ± 17.7 −16.9 ± 17.6 16.2 [2.3, 0.62 [0.09, Likely
(91%)
Pre- −7.7 ± 15.4 −8.7 ± 15.6 1.0 [−9.8, 0.04 Unclear
72 h 13.1] [−0.42,
0.51]
Isometric Pre- −10.3 ± 13.4 −11.7 ± 20.7 1.6 [−10.5, 0.06 Unclear
peak torque 24 h 15.3] [−0.40,
(Nm) 0.51]
Pre- −5.9 ± 15.1 −9.4 ± 24.1 3.8 [−10.2, 0.13 Unclear
48 h 19.9] [−0.38,
0.65]
Pre- −21.8 ± 145.1 −8.2 ± 26.1 −14.8 −0.58 Unclear
72 h [−50.2, [−2.51,
45.7] 1.35]
Analyses were adjusted to baseline grand means for each outcomes
a
Compared with high-intensity passive static stretching, low-intensity passive static

stretching had a very likely moderate beneficial effect on eccentric peak torque from
baseline to 24 and 72 h following an unaccustomed eccentric exercise. The effect
from baseline to 48 h was possibly small beneficial effect (see Table 5.2).
Results 173
Fig. 5.5 Eccentric peak

torque (raw)
Fig. 5.6 Histogram of eccentric peak torque difference
Compared with control condition, low-intensity passive static stretching had a very
likely moderate and likely small beneficial effect on eccentric peak torque from
baseline to 48 and 72 h, respectively. The effect from baseline to 24 h was unclear
(see Table 5.3).
From baseline to 24 h post-unaccustomed eccentric exercise, high-intensity passive

static stretching had a likely small harmful effect on eccentric peak torque, but a
likely moderate beneficial effect from baseline to 48 h, compared with control. The
effect from baseline to 72 h was unclear (see Table 5.4).
Fig. 5.7 Eccentric peak torque 72 vs. 0 h (scatter plot)
Fig. 5.8 Isometric peak

torque (raw). *LIS
low-intensity passive static
stretching, HIS high-
intensity passive static
stretching
Isometric Peak Torque
RMANOVA
Although no significant difference was observed between time x condition interac-

tion (p = 0.185) (Table 5.1), a significant main effect of time (p < 0.001) was
observed. Post hoc tests showed that values were lower at 24 h compared with base-
line (p < 0.001), higher at 48 compared with 24 h (p = 0.036), and at 72 compared
Results 175
Fig. 5.9 Histogram of isometric peak torque difference
Fig. 5.10 Isometric peak torque 72 vs. 0 h (scatter plot)
with both 24 (p < 0.001) and 48 (p = 0.014), compared with 24 h (p < 0.01)
(Table 5.1, Fig. 5.8). Both the histogram (Fig. 5.9) and scatter plot (Fig. 5.10) sug-
gest that low-intensity passive static stretching was affiliated with improvements in
isometric peak torque (increase in values) compared to both high-intensity passive
static stretching and no stretching (control).
The effect of low-intensity passive static stretching compared with high-intensity

passive static stretching was trivial from baseline to 24 h, possibly small (beneficial)
from baseline to 48 h, and unclear from baseline to 72 h post-unaccustomed eccen-
tric exercise (Table 5.2).
Compared with control, low-intensity passive static stretching resulted in possibly

and likely small beneficial effects on isometric peak torque from baseline to 48 and
72 h, respectively. From baseline to 24 h, the effects were unclear (Table 5.3).
The effects of high-intensity passive static stretching compared with control were
unclear for comparisons between each time point compared with baseline
(Table 5.4).
Discussion
To our knowledge, this is the first study that investigated in a randomised design the
effects of different passive static stretching intensities following an unaccustomed
eccentric exercise on perceived muscle soreness and muscle function. Our results
suggest that low-intensity passive static stretching may lower perceived muscle
soreness and improve muscle function (eccentric and isometric peak torque) largely
compared with high-intensity passive static stretching and control.
Studies on stretching and DOMS following unaccustomed eccentric exercise
have investigated the timing of stretching (i.e. before, after, or before and after exer-
cise) and whether stretching produces reductions in muscle soreness (Gulick,
Kimura, Sitler, Paolone, & Kelly, 1996; Herbert et al., 2011; High et al., 1989;
Johansson et al., 1999; Lund et al., 1998; Wessel & Wan, 1994). To our knowledge,
only Smith et al. (1993) investigated the effects of stretching on the induction of
DOMS, evaluating a bout of static versus ballistic stretching of similar intensity and
duration on the induction of DOMS over a 5-day period. This study revealed that
static stretching was responsible for inducing significantly more DOMS than bal-
listic stretching. Our study adds to the body of knowledge in this field, suggesting
that low-intensity passive static stretching (30–40% of maximum) is at least very
likely effective compared with the other two conditions in reducing perceived mus-
cle soreness over 72 h, possibly due to the difference in the magnitude of the stretch
between the conditions. In line with our observations, Behm and Kibele (2007) sug-
Discussion 177
gest that stretches to a maximal point of discomfort, as well as submaximal stretch-

ing greater than 50% of point of discomfort, are responsible for muscle damage,
similar to the effects seen with the eccentric-induced damage associated with
DOMS. Another potential reason for these likely beneficial effects of low-intensity
passive static stretching is the time that the stretching was held (i.e. 60 s). Our find-
ings are in line with a study of individuals ages 65 years or greater, where low-
intensity stretching held for 60 s was found to be more effective for increasing knee
extension ROM compared to 30 s, 15 s, or control (Feland, Myrer, Schulthies,
Fellingham, & Measom, 2001). In addition, a clear dose-response effect for stretch
duration has been reported, suggesting that stretches held greater than 60 s are more
likely to impair performance (Kay & Blazevich, 2012). Therefore, despite the dif-
ferent participant samples utilised in these investigations and our study, taken col-
lectively, these observations suggest that the time passive static stretching held may
have implications on perceived muscle soreness. Further research is required to
elucidate these phenomena.
With regard to muscle function, several studies and systematic reviews have
reported that stretching can induce a decrease in strength or power output for iso-
lated muscle groups following an unaccustomed eccentric exercise (Behm,
Blazevich, Kay, & Mchugh, 2016; Behm, Button, & Butt, 2001; Behm & Chaouachi,
2011; Church, Wiggins, Moode, & Crist, 2001; Cramer et al., 2004; Cramer et al.,
2005; Kay & Blazevich, 2012; Kokkonen, Nelson, & Cornwell, 1998; Marek et al.,
2005; Nelson & Kokonnen, 2001; Power, Behm, Farrel, Carroll, & Young, 2004;
Young & Elliot, 2001). For instance, a systematic review highlighted a clear dose-
response effect for stretch duration, with longer held acute static stretches (≥ 60 s)
being more likely to elicit performance impairments compared to shorter duration
stretching (<60 s) (Kay & Blazevich, 2012). In line with this, a recent study revealed
that longer stretch durations elicit performance impairments, which may have
implications for athletic performance and clinical outcomes (Behm et al., 2016).
Whereas some studies tested participants immediately after stretching (Cramer
et al., 2005; Kokkonen et al., 1998; Marek et al., 2005), a study that retested for
explosive performance (long jump) and repeated sprint ability 24 h post-stretching
concluded that static stretching continued to have a negative effect on performance
(Haddad et al., 2014). However, it is noteworthy that the available studies did not
take into account the intensity of the stretching exercises. To investigate the poten-
tial role of stretching intensity, Behm and Kibele (2007) observed that to achieve
maximum jump height, athletes should perform static stretching of an intensity less
than 50% of point of discomfort prior to performance. In line with this finding, our
data show that low-intensity passive static stretching resulted in small-to-moderate
beneficial effects on eccentric peak torque compared to both high-intensity passive
static stretching and control. Our findings for isometric peak torque were less con-
sistent, with low-intensity passive static stretching resulting in beneficial effects
over control (baseline—72 h) but not high-intensity passive static stretching.
However, the observed improvements in eccentric peak torque over the three con-
secutive days with low-intensity passive static stretching suggest an increased level
of recovery, given that a decrease in maximal muscle torque is considered the best
index of muscle damage (Paschalis, Nikolaidis, et al., 2007).
Conclusion
Compared to high-intensity passive static stretching and no stretching, low-intensity

passive static stretching results in small-to-moderate beneficial effects on perceived
muscle soreness and recovery of muscle function post-unaccustomed eccentric exer-
cise. Since soreness associated with DOMS affects athletic performance, a decrease
in perceived muscle soreness may aid in the restoration of maximal muscle function
and an increased adherence to regular exercise with nonathletes. Future studies
should employ larger populations, use supervised stretching, and assess both indirect
measures [eccentric and isometric peak torque, blood biomarkers (creatine kinase,
hsCRP), inflammatory cytokines (IL-1β, TNF-α, and IL-6), and subjective soreness
responses], and direct measures, such as examination of muscle samples (biopsies),
to determine muscle damage following unaccustomed eccentric exercise.
Permission
Some of the material in this chapter has been taken from:

Apostolopoulos, N. C., Lahart, I. M., Plyley, M. J., Taunton, J., Nevill, A. M.,
Koutedakis, Y., … & Metsios, G. S. (2018). The effects of different passive static
stretching on recovery from unaccustomed eccentric exercise–a randomized
controlled trial. Applied Physiology Nutrition and Metabolism, 43(8), 806–815.
Copyright © 2018 by Canadian Science Publishing (CSP) or its licensors.
Reprinted by permission of CSP press.
References
& Health Sciences, 2, 399–406.
Apostolopoulos, N. (2010). Microstretching-a practical approach for recovery and regeneration.
Armstrong, R. B. (1984). Mechanisms of exercise-induced delayed onset muscular soreness: a
brief review. Medicine and Science in Sports and Exercise, 16, 529-538.
Batterham, A. M., & Hopkins, W. G. (2005). Making meaningful inferences about magnitudes.
Sport Science, 2002, 6–13.
Bauer, J., Lengyel, G., Bauer, T. M., Acs, G., & Gerok, W. (1989). Regulation of interleukin-6
receptor expression in human monocytes and hepatocytes. FEBS Letters, 249, 27–30.
Beaton, L. J., Allan, D. A., Tarnopolsky, M. A., Tiidus, P., & Phillips, D. R. (2002a). Contraction
induced muscle damage is unaffected by vitamin E supplementation. Medicine and Science in
Beaton, L. J., Tarnopolsky, M. A., & Phillips, S. M. (2002b). Contraction-induced muscle damage
in humans following calcium channel blocker administration. The Journal of Physiology, 544,
849–859.
References 179
Behm, D. G., & Chaouachi, A. (2011). A review of the acute effects of static and dynamic stretch-
ing on performance. European Journal of Applied Physiology, 11, 2633–2651.
Behm, D. G., Blazevich, A. J., Kay, A. D., & Mchugh, M. (2016). Acute effects of muscle stretch-
ing on physical performance, range of moiton, and injury incidence in healthy active individu-
als: a systematic review. Applied Physiology, Nutrition, and Metabolism, 41, 1–11.
Behm, D. G., Button, D. C., & Butt, J. C. (2001). Factors affecting force loss with prolonged
stretching. Canadian Journal of Applied Physiology, 26, 261–272.
Byrnes, W. C., & Clarkson, P. M. (1986). Delayed onset muscle soreness and training. Clinics in
Castell, J. V., Andus, T., Kunz, D., & Heinrich, P. C. (1989). Interleukin-6 the major regulator of
acute-phase protein synthesis in man and rat. Annals of the New York Academy of Sciences,
557, 87–101.
Castell, J. V., Gomez-Lechion, M. J., David, M., Hirano, T., Kishimoto, T., & Heinrich, P. C.
(1988). Recombinant human interleukin-6 (Il-6/Bsf-2/Hsf) regulates the synthesis of acute
phase proteins in human hepatocytes. Feb Letters, 232, 347–350.
Cheung, K., Hume, P., & Maxwell, L. (2003). Delayed onset muscle soreness: treatment strategies
Church, J. B., Wiggins, M. S., Moode, F. M., & Crist, R. (2001). Effect of warm-up and flexibility
treatments on vertical jump performance. Journal of Strength and Conditioning Research, 15,
332–336.
Clarkson, P. M., Nosaka, K., & Braun, B. (1992). Muscle function after exercise-induced muscle
damage and rapid adaptation. Medicine and Science in Sports and Exercise, 24, 512–520.
Cramer, J. T., Hough, T. J., Johnson, G. O., Miller, J. M., Coburn, J. W., & Beck, T. W. (2004).
The effects of static stretching on peak torque in women. Journal of Strength and Conditioning
Research, 18, 176–182.
Cramer, J. T., Hough, T. J., Weir, J. P., Johnson, G. O., Coburn, J. W., & Beck, T. W. (2005). The
acute effects of static stretching on peak torque, mean power output, electromyography, and
mechanomyography. European Journal of Applied Physiology, 93, 540–539.
contractions: conditions of occurrence and prevention. Physical Therapy, 73, 911–921.
Feland, J. B., Myrer, J. W., Schulthies, S., Fellingham, G., & Measom, G. (2001). The effect of
duration of stretching of the hamstring muscle group for increasing range of motion in people
aged 65 years or older. Physical Therapy, 81, 1110–1117.
Frenette, J., Cai, B., & Tidball, J. G. (2000). Complement activation promotes muscle inflamma-
tion during modified muscle use. The American Journal of Pathology, 156, 2103–2110.
Friden, J., & Lieber, R. L. (2001). Eccentric exercise-induced injuries to contractile and cytoskel-
etal muscle fibre compnents. Acta Physiologica Scandinavica, 171, 321–326.
Friden, J., Sjostrom, M., & Ekblom, B. (1983). Myofibrillar damage following intense eccentric
exercise in man. International Journal of Sports Medicine, 4, 170–176.
Gulick, D. T., Kimura, I. F., Sitler, M., Paolone, A., & Kelly, J. D. (1996). Various treatment tech-
niques on signs and symptoms of delayed onset muscle soreness. Journal of Athletic Training,
31, 145–152.
Gullick, D. T., & Kimura, I. F. (1996). Delayed onset muscle soreness: what is it and how do we
treat it? Journal of Sport Rehabilitation, 5, 234–243.
Haddad, M., Dridi, A., Chtara, M., Chaouachi, A., Wong, D. P., Behm, D., et al. (2014). Static
stretching can impair explosive performance for at least 24 hours. Journal of Strength and
Hasson, S., Barnes, W., Hunter, M., & Williams, J. (1989). Therapeutic effects of high speed volun-
tary muscle contractions on muscle soreness and muscle performance. Journal of Orthopaedic
& Sports Physical Therapy, 10, 499–507.
Hasson, S. M., Daniels, J. C., Divine, J. G., Niebuhr, B. R., Richmond, S., Stein, P. G., et al. (1993).
Effect of ibuprofen use on muscle soreness, damage, and performance: a preliminary investiga-
tion. Medicine and Science in Sports and Exercise, 25, 9–17.
Herbert, R. D., De Noronha, M., & Kamper, S. J. (2011). Stretching to prevent or reduce muscle
soreness after exercise. Cochrane Database of Systematic Reviews, 7, 1–47.
Hertel, J. (1997). The role of nonsteroidal anti-inflammatory drugs in the treatment of acute soft
tissue injuries. Journal of Athletic Training, 32, 350–358.
High, D. M., Howley, E. T., & Franks, B. D. (1989). The effects of static stretching and warm-up
on prevention of delayed onset muscle soreness. Research Quarterly for Exercise and Sport,
60, 357–361.
Hopkins, W. G. (2002). Probabilities of clinical or practical significance. Sportscience, 6(201), 16.
Hopkins, W. G. (2006). Spreadsheets for analysis of controlled trials, with adjustment for a subject
characteristic. Sportscience, 10, 46–50.
Hopkins, W. G. (2007). A spreadsheet for deriving a confidence interval, mechanistic inference
and clinical inference from a p value. Sportscience, 11, 16–20.
Janssen, E., Kuipers, H., Vertsappen, F., & Costill, D. (1983). Influence of anti-inflammatory drugs
on muscle soreness. Medicine and Science in Sports and Exercise, 15, 165.
Johansson, P. H., Lindstrom, L., Sundelin, G., & Lindstrom, B. (1999). The effects of preexercise
stretching on muscular soreness, tenderness and force loss following heavy eccentric exercise.
Scandinavian Journal of Medicine & Science in Sports, 9, 219–225.
Kanda, K., Sugama, K., Hayashida, H., Sakuma, J., Kawakami, Y., Miura, S., et al. (2013).
Eccentric exercise-induced delayed-onset muscle soreness and changes in markers of muscle
damage and inflammation. Exercise Immunology Review, 19, 72–85.
Kay, A. D., & Blazevich, A. J. (2012). Effect of acute static stretch on maximal muscle perfor-
mance: a systematic review. Medicine & Science in Sports & Exercise, 44, 154–164.
Kokkonen, J., Nelson, A. G., & Cornwell, A. (1998). Acute muscle stretching inhibits maximal
strength performance. Research Quarterly for Exercise and Sport, 69, 411–415.
Kushner, I., & Rzewnicki, D. L. (1994). The acute phase response: general aspects. Baillière's
Clinical Rheumatology, 8, 513–530.
Lightfoot, T. J., Char, D., Mcdermott, J., & Goya, C. (1997). Immediate post exercise massage
does not attenuate delayed onset muscle soreness. Journal of Strength and Conditioning
Research, 11, 119–124.
Lund, H., Vestergaard-Poulsen, P., Kanstrup, I.-L., & Sejrsen, P. (1998). The effect of passive
stretching on delayed onset muscle soreness, and other detrimental effects following eccentric
exercise. Scandinavian Journal of Medicine & Science in Sports, 8, 216–221.
Macintyre, D. L., Reid, W. D., Lyster, D. M., & Mckenzie, D. C. (2000). Different effects of
strenuous eccentric exercise on the accumulation of neutrophils in muscle in women and men.
Macintyre, D. L., Reid, W. D., & Mckenzie, D. C. (1995). The inflammatory response to muscle
Malm, C., Nyberg, P., Engstrom, M., Sjodin, B., Lenkei, R., & Ekblom, B. (2000). Immunological
changes in human skeletal muscle and blood after eccentric exercise and multiple biopsies. The
Marek, S. M., Cramer, J. T., Fincher, A. L., Massey, L. L., Danglemeier, S. M., Purkayastha, S.,
et al. (2005). Acute effects of static and proprioceptive neuromuscular facilitation on muscle
strength and power output. Journal of Athletic Training, 40, 94–103.
Maxwell, S., Kohl, S., Watson, A., & Belnave, R. J. (1988). Is stretching effective in the preven-
tion of or amelioration of delayed onset muscle soreness. Australian Journal of Science and
Medicine in Sport, 20, 22.
Nelson, A. G., & Kokonnen, J. (2001). Acute ballistic muscle stretching inhibits maximal strength
performance. Research Quarterly for Exercise and Sport, 72, 415–419.
Newham, D. J., Jones, D. A., & Clarkson, P. M. (1987). Repeated high-force eccentric exercise:
effects on muscle and damage. Journal of Applied Physiology, 63, 1381–1387.
Newham, D. J., Mills, K. R., Quigley, B. M., & Edwards, R. H. T. (1983). Pain and fatigue after
concentric and eccentric contractions. Clinical Science, 64, 55–62.
References 181
Nosaka, K., & Clarkson, P. M. (1992). Relationship between post exercise plasma ck elevation and
muscle mass involved in the exercise. International Journal of Sports Medicine, 13, 471–475.
Paschalis, V., Koutedakis, Y., Jamurtas, A. Z., Mougios, V., & Baltzopoulos, V. (2005). Equal vol-
umes of high and low intensity of eccentric exercise in relation to muscle damage and perfor-
mance. Journal of Strength and Conditioning Research, 19, 184–188.
Paschalis, V., Giakas, G., Baltzopoulos, V., Jamurtas, A. Z., Theoharis, V., Kotzamanidis, C., et al.
(2007a). The effects of muscle damage following eccentric exercise on gait biomechanics. Gait
& Posture, 25, 236–242.
Paschalis, V., Nikolaidis, M. G., Fatouros, I. G., Giakas, G., Koutedakis, Y., Karatzaferi, C., et al.
(2007b). Uniform and prolonged changes in blood oxidative stress after muscle-damaging
exercise. In Vivo, 21, 877–883.
Peake, J. M. (2002). Exercise-induced alterations in neutrophil degranulation and respiratory burst
activity: possible mechanisms of action. Exercise Immunology Review, 8, 49–100.
Peake, J. M., Nosaka, K., & Suzuki, K. (2005). Characterization of inflammatory responses to
eccentric exercise in humans. Exercise Immunology Review, 11, 64–85.
Power, K., Behm, D., Farrel, C., Carroll, M., & Young, W. (2004). An acute bout of static stretch-
ing: effects on force and jumping performance. Medicine and Science in Sports and Exercise,
36, 1389–1396.
Robson, P. J., Blannin, A. K., Allan, D. A., Walsh, N. P., Castell, L. M., & Gleeson, M. (1999).
Effects of exercise intensity, duration and recovery on in vitro neutrophil function in male ath-
letes. International Journal of Sports Medicine, 20, 128–135.
Rowlands, A. V., Eston, R. G., & Tilzey, C. (2001). Effect of stride length manipulation on
symptoms of exercise-induced muscle damage and the repeated bout effect. Journal of Sports
Sciences, 19, 333–340.
Shizuo, A., Hirano, T., Taga, T., & Kishimoto, T. (1990). biology of multifunctional cytokines: Il-6
and related molecules (Il-1 and tnf). The FASEB Journal, 4, 2860–2867.
Smith, L. L. (1991). Acute inflammation: the underlying mechanism in delayed onset muscle
Smith, L.L., Brunetz, M. H., Chenier, T. C., McCammon, M. R., Houmard, J. A., Franklin, M. E.,
& Israel, R. G. (1993). The effects of static and ballistic stretching on delayed onset muscle
soreness and creatine kinase. Research Quarterly for Exercise and Sport, 64, 103–107.
Stupka, N., Tarnopolsky, M. A., Yardley, N. J., & Phillips, S. M. (2001). Cellular adaptation to
repeated eccentric exercise-induced muscle damage. Journal of Applied Physiology, 91,
1669–1678.
Tiidus, P., & Ianuzzo, C. (1983). Effects of intensity and duration of muscular exercise on delayed
onset muscle soreness and serum enzymes activities. Medicine and Science in Sports and
Exercise, 15, 461–465.
Vasudevan, S. V. (1993). Impairment, disability and functional capacity assessment. New York:
The Guildford Press.
Weber, M. D., Servedio, F. J., & Woodall, W. R. (1994). The effects of three modalities on delayed
onset muscle soreness. Journal of Orthopaedic & Sports Physical Therapy, 20, 236–242.
Wessel, J., & Wan, A. (1994). Effect of stretching on the intensity of delayed-onset muscle sore-
ness. Clinical Journal of Sport Medicine, 4, 83–87.
Worrel, T. W., Perrin, D. H., & Denegar, C. R. (1989). The influence of hip position on quadriceps
and hamstring peak torque and reciprocal muscle group ratio values. Journal of Orthopaedic
and Sports Physical Therapy, 11, 104–107.
Young, W., & Elliot, S. (2001). Acute effects of static stretching, proprioceptive neuromuscular
facilitation stretching, and maximum voluntary contractions on explosive force production and
jumping performance. Research Quarterly for Exercise and Sport, 72, 273–279.
Chapter 6
Summary Discussion
Introduction
The magnitude of the force applied during a stretching exercise is defined as the
intensity of the stretch (Jacobs & Sciacia, 2011). The three studies of this manu-
script specifically observed how stretching intensity, in the form of either a low-,
moderate-, or high-intensity passive static stretch, influences the inflammatory
response. To investigate these effects, hsCRP, and the pro-inflammatory cytokines
IL-1β, TNF-α, and IL-6, involved in the regulation of the immune response, were
measured in both studies 1 and 2. To our knowledge, these were the first to measure
effects of passive static stretching intensity through the use of blood biomarkers for
inflammation in humans. With study 3, concern was to investigate how low- and
high-intensity passive static stretching influences perceived muscle soreness and
muscle function. Since soreness associated with DOMS affects athletic perfor-
mance, a strategy introduced to diminish the severity of DOMS sooner aids in res-
toration of muscle function (Cheung, Hume, & Maxwell, 2003). For nonathletes,
early alleviation from symptoms associated with DOMS postexercise increases
adherence to regular exercise (Smith et al., 1994). A brief summary of the findings
for all three strudies is found in the table below (Table 6.1).
Acute Inflammatory Response to Stretching
The aim of this study was to examine whether high-intensity passive static stretch-
ing (IS) was responsible for causing acute inflammation, as measured by serum-
based inflammation biomarkers. Twelve healthy recreationally active males, acting
as their own control, participated in a randomised crossover trial. During the stretch-
ing intervention, both the right and left hamstrings, glutes, and quadriceps muscles
were exposed to a high-intensity passive static stretch by the same therapist,

184
Table 6.1 Summary of studies

Stretch Results
intensity Inflammation
Study n Design L M H hsCRP IL-1β IL-6 TNF-α Discussion
1 12 Randomised within A significant increase in the concentration of hsCRP was observed
subject crossover comparing high-intensity passive static stretching (IS) to control.
trial (IS vs. con) This was confirmed with effect sizes looking at between
conditions. Within comparisons also revealed a small increase in
hsCRP for high-intensity passive static stretching vs. control
conditions
2 11 Randomised within Both low- (30% maximum ROM) and medium (60%maximum
subject crossover ROM) intensity passive static stretching did not elicit an
trial (30, 60, & inflammatory response, with concentration for hsCRP. A
90% maximum pronounced inflammatory response was observed comparing
ROM) 30–90 and 60–90% maximum ROM; however, caution is needed
since a discrepancy with values for 90% was detected at baseline.
In addition, our secondary analysis revealed that for every
increase in % maximum ROM, there was a moderate increase in
hsCRP
3 30 Randomised Low-intensity passive static stretching is likely to result in
clinical trial (LIS, small-to-moderate beneficial effects on perceived muscle soreness
HIS and con) and recovery of muscle function (eccentric and isometric peak
torque) post-unaccustomed eccentric compared with high-
intensity passive static stretching and control (no stretching)
n number of participants, L low, M medium, H high-intensity passive static stretching, hsCRP high-sensitivity C-reactive protein, IL-1β interleukin-1β, IL-6
interleukin-6, TNFα tumour necrosis factor alpha
6 Summary Discussion
Stretch Intensity Vs. Inflammation: Is there a Dose-Dependent Association? 185
standardising the stretching intensity for all participants. Using a numerical rating
scale, stretching intensity was maintained at a level rated as discomfort/pain (8 out
of 10), with each muscle group stretched for 3 × 60 s for a total of 18 min. During
the control intervention, participants rested for the equivalent amount of time. The
blood was collected pre-, immediately post, and 24 h post-interventions and anal-
ysed for hsCRP (primary outcome), as well as for IL-1β, TNFα, and IL-6. To mea-
sure levels of C-reactive protein, hsCRP, which is more sensitive than standard tests
for C-reactive protein, was used to evaluate inflammation. Compared to control,
hsCRP increased significantly immediately post and 24 h post for time (p = 0.005)
and time x condition (p = 0.006). However, since the half-lives for the pro-
inflammatory cytokines (IL-1β, and, TNF-α and IL -6) were missed, no data was
available. Similar to studies that measured hsCRP in rugby (Cunniffe et al., 2011,
Lund, Hurst, Tyrell, & Thompson, 2011), plyometric exercise (Chatzinikolaou
et al., 2010), and long duration running (Kim, Lee, & Kim, 2007; Mooren et al.,
2006), a significant increase in hsCRP (14-fold) was observed 24 h post, with high-
intensity passive static stretching. A study measuring for hsCRP in the latter half of
a 200 km ultramarathon race observed a 23-fold increase (Kim et al., 2007).
Although the claim is not being made that high-intensity passive static stretching is
similar to long duration or high-intensity activities, the sharp increase in hsCRP is
interesting, possibly a reflection of the amount of muscle mass being recruited
(Febbraio, Hiscock, Sacchetti, Fischer, & Pedersen, 2004). Activities using large
masses of muscle such as a 160 km triathlon (Taylor et al., 1987), and 6-days
ultramarathon race (Fallon, 2001), observed an increase in C-reactive protein,
50.8 mg/L and 37.5 mg/L, respectively. More research needs to be conducted. In
addition, effect sizes between and within groups suggest that high-intensity passive
static stretching was associated with an increase in hsCRP concentration compared
to the control condition.
tretch Intensity Vs. Inflammation: Is there a Dose-Dependent

S
Association?
To our knowledge, study 2, a continuation of study 1, was the first to report the
effects of three different stretching intensity interventions on an inflammatory bio-
marker. This randomised, within subject design crossover trial, conducted over 3
consecutive weeks, consisted of 11 healthy recreationally active males. Each par-
ticipant performed and completed the three passive static stretching intensity condi-
tions calculated at 30, 60, and 90% of the maximum ROM of the right hamstring
muscle. Each percentage was representative of a low- (30%), medium- (60%), and
high- (90%) stretching intensity. Using a kinetic dynamometer, the right hamstring
muscle was placed at the appropriate percentage of maximum ROM and stretched
at the respective intensity for five repetitions at 60 s, followed by a 10-s rest between
each repetition. The blood was collected pre-, immediately post, and at 24 h
186 6 Summary Discussion
post-interventions and was analysed for hsCRP. Based on the results for hsCRP in
relation to the % maximum ROM, stretching intensity between 30 and 60% of max-
imum ROM did not induce significant increases in the concentration of hsCRP at
24 h (p > 0.05), suggesting that these levels do not elicit a significant systemic
inflammatory response. Comparing 30–90 and 60–90% maximum ROM, a signifi-
cant increase in the concentration level of hsCRP was observed for 30–90 (p = 0.004)
and 60–90 (p = 0.034). However, since an anomaly was observed at baseline for
90% maximum ROM, caution should be exercised. The secondary analysis revealed
that for every increase in percentage maximum ROM, there was a moderate increase
in hsCRP. In conclusion, the results reported from this study provide evidence for
the association of inflammation and stretching intensity.
he Effects of Different Passive Static Stretching Intensities

T
on Perceived Muscle Soreness and Muscle Function Recovery
Following Unaccustomed Eccentric Exercise: A Randomised
Trial
With study 3, the effects of passive static stretching intensity on recovery from
unaccustomed eccentric exercise of the right knee extensors was investigated. Thirty
recreationally active males were randomly allocated into three groups: low intensity
(30–40% maximum perceived stretch), high intensity (70–80% maximum perceived
stretch), and no stretching (control), with each condition consisting of ten partici-
pants. Both stretching groups performed three sets of passive static stretching exer-
cises for a duration of 60 s each for hamstrings, hip flexors, and quadriceps, over 3
consecutive days, following unaccustomed eccentric exercise. Muscle function
(eccentric and isometric peak torque) was measured before (baseline) and after (24,
48, and 72 h) unaccustomed eccentric exercise. Perceived muscle soreness scores
were collected immediately (time 0) and after 24, 48, and 72 h postexercise.
Statistical time x condition interactions was observed only for eccentric peak torque
(p = 0.008). Magnitude-based inference analyses revealed low-intensity passive
static stretching had most likely, very likely, or likely beneficial effects on perceived
muscle soreness (48–72 h and 0–72 h) and eccentric peak torque (baseline-24 and
baseline-72 h), compared with high-intensity stretching. Compared with control,
low-intensity passive static stretching had very likely or likely beneficial effects on
perceived muscle soreness (0–24 and 0–72 h), eccentric peak torque (baseline-48 h
and baseline-72 h), and isometric peak torque (baseline-72 h). High-intensity
stretching had likely beneficial effects on eccentric peak torque (baseline-48 h) but
likely harmful effects eccentric peak torque (baseline-24 h) compared with control.
Therefore, low-intensity passive static stretching is likely to result in small-to-
moderate beneficial effects on perceived muscle soreness and recovery of muscle
function (eccentric and isometric peak torque) post-unaccustomed eccentric exer-
cise, compared with high intensity or no stretching.
References 187
References
Chatzinikolaou, A., Fatouros, I., Gourgoulis, V., Avloniti, A., Jamurtas, A. Z., Nikolaidis, M. G.,
et al. (2010). Time course of changes in performance and inflammatory responses after acute
plyometric exercise. Journal of Strength and Conditioning Research, 24, 1389–1398.
Cunniffe, B., Hore, A. J., Whitcombe, D. M., Jones, K. P., Davies, B., & Baker, J. S. (2011).
Immunoendocrine responses over a three week international rugby union series. The Journal
of Sports Medicine and Physical Fitness, 51, 329–338.
Fallon, K. E. (2001). The acute phase response and exercise: The ultramarathon as prototype exer-
cise. Clinical Journal of Sport Medicine, 11, 38–43.
Febbraio, M. A., Hiscock, N., Sacchetti, M., Fischer, C. P., & Pedersen, B. K. (2004). Interleukin-6
is a novel factor mediating glucose homeostasis during skeletal muscle contraction. Diabetes,
53, 1643–1648.
Kim, H. J., Lee, Y. H., & Kim, C. K. (2007). Biomarkers of muscle and cartilage damage and
inflammation during a 200 km run. European Journal of Applied Physiology, 99, 443–447.
Lund, A. J., Hurst, T. L., Tyrell, R. M., & Thompson, D. (2011). Markers of chronic inflammation
with short-term changes in physical activity. Medicine and Science in Sports and Exercise, 43,
578–583.
Mooren, F. C., Lechtermann, A., Fobker, M., Brandt, B., Sorg, C., Volker, K., et al. (2006). The
response of the novel pro-inflammatory molecules S100a8/A9 to exercise. International
Smith, L. L., Keating, M. N., Holbert, D., Spratt, D. J., Mccammon, M. R., Smith, S. S., et al.
(1994). The effects of athletic massage on delayed onset muscle soreness, creatine kinase, and
neutrophil count: A preliminary report. Journal of Orthopaedic & Sports Physical Therapy,
19, 93–99.
Taylor, C., Rogers, G., Goodman, C., Baynes, R. D., Bothwell, T. H., Bezwoda, W. R., et al.
(1987). Hematologic, iron-related, and acute-phase protein responses to sustained strenuous
exercise. Journal of Applied Physiology, 62, 464–469.
Chapter 7
Limitations
Limitations of Studies (Table 7.1)
Blood Collection
A major limitation regarding studies one and two was the design for the collection
of blood samples. Although hsCRP was measured in these studies, and study 1 did
measure IL-1β, IL-6, and TNFα, the design for collecting the blood samples missed
the half-life of the pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α). In the
literature, the half-life for TNF-α, IL-1β, and IL-6 are approximately 18.2 min
(Oliver et al., 1993), 2.5 h (Hazuda, Lee, & Young, 1988), and 2–4 h (Marino &
Giotta, 2008; Pedersen, 2000; Rohde, Maclean, Richter, Kiens, & Pedersen, 1997),
respectively. Since the immune response is regulated by cytokines released by
immune cells (neutrophils, monocytes, and macrophages) in response to stimulus
(Lacy & Stow, 2011), these pro-inflammatory proteins are part of a cytokine net-
work in which the expression of one is influenced by another. In specific, IL-1β and
TNF-α are potent inducers of IL-6, with IL-6 inversely regulating TNF-α expression
(Akira, Hirano, Taga, & Kishimoto, 1990). The main physiological function of IL-6
is the induction of the hepatic acute phase response characterised by the increased
expression of acute phase proteins, specifically C-reactive protein (Bauer, Lengyel,
Bauer, Acs, & Gerok, 1989; Gabay & Kushner, 1999). Although blood collection
failed to measure the half-life for IL-6, the literature suggests that increased expres-
sion of C-reactive protein is due to a relevant increase in IL-6. The absence of IL-6
prevents proper recovery (Mcfarland-Mancini et al., 2010) since it is necessary for
the resolution of inflammation as observed in mice deficient in IL-6 (IL-6−/−) (Kopf
et al., 1994). Although the IL-6−/− mice did not express any developmental abnor-
malities, they were unable to generate an acute phase response (Kopf et al., 1994).
The induction of IL-6 due to injury or infection is believed to be a very important
in vivo distress signal coordinating the activities of the hepatocytes, macrophages,
and lymphocytes (Kopf et al., 1994). Since the relationship between IL-6 and

Table 7.1 Summary of studies and scheduled time periods for blood collection
190
Stretching
intensity Limitations
Inflammation Blood collection periods
Study L M H C hsCRP IL-1β IL-6 TNF-α Pre Post 24 h Post
1 • Blood collection missed half-lives for IL-1β,
IL-6, and TNF-α
• Compliance
• Nutrition
• Mental stress
• Age (no elderly)
2 • IL-6 was not measured
• Methodological (intensity level) 90% vs. 80%
(study 1)
• Methodological (repetitions) 5 vs. 3 sets (studies
1 and 3)
• Compliance
• Nutrition
• Mental stress
3 • Methodological (range of intensity) 30–40% and
70–80% vs. 80% (study 1), 30% (study 2)
• Compliance
• Nutrition
• Mental stress
• Lack of sufficient statistical power
• Data from isokinetic dynamometry—
quantification of unaccustomed eccentric exercise
L low, M moderate, H high-intensity passive static stretching, C control, hsCRP high sensitivity C-reactive protein, IL-1β interleukin-1 beta, IL-6 interleukin-6,
TNFα tumour necrosis factor alpha
7 Limitations
Limitations of Studies 191
C-reactive protein represents the body’s mechanism for resolving the acute inflam-
matory response, the observed increase in hsCRP was due to relevant changes in
IL-6 (Akira et al., 1990; Fuller & Grenett, 1989; Gruys, Toussaint, Niewold, &
Koopmans, 2005; Samols, Agrawal, & Kushner, 2002; Streetz, Wustefeld, Klein,
Manns, & Trautwein, 2001). Therefore, future studies would collect blood at the
appropriate times (half-lives) for the pro-inflammatory cytokines to determine the
relevant relationship between these cytokines and the acute inflammatory response,
in response to low- and high-intensity passive static stretching, and no stretching.
Methodology
The methodological progression regarding both stretching intensity and the number
of sets performed per stretching exercise may be considered as another limitation.
Participants in study 1 were stretched at an intensity level of 80% of their maximum
perceived exertion (slight pain and discomfort). In study 2, despite the focus being
on the association between various stretching intensities [low (30 %), medium
(60%), and high (90%)] and the inflammatory response, use of 80 instead of 90%
intensity for high-intensity passive static stretching would have ensured continuity.
With study 3, a range of intensity was introduced for low- (30–40%) and high (70–
80 %)-intensity passive static stretching. Whereas this zone represents a method-
ological change, it should be emphasised that it provided a leeway for the
interpretation of the stretch sensation. Unlike the other two studies where the inves-
tigator and the participants had direct contact, and monitoring was possible, ensur-
ing maintenance of the proper intensity, with study 3, the stretching groups
performed the exercises at home before sleep. The use of a range, accompanied with
a description of the stretch sensation (i.e., gentle, warm, pain, discomfort), con-
veyed the magnitude of the stretch needed. According to psychophysical laws, this
is a form of “magnitude production”, in which the range provided to the participant
allows for the best adjustments in order to produce a match (Stevens, 1971).
Although the number of sets per stretching exercise was different (i.e. three sets
for study 1 and 3, and five for study 2), suggesting a discontinuity, a study observed
that the first four stretches of a repeated stretching of the MTU, to 10% beyond rest-
ing length and to a set tension, resulted in the greatest changes (Taylor, Dalton,
Seaber, & Garrett, 1990). This study concluded that little alteration of the MTU
occurs after four repetitive stretches, suggesting that this is the minimum amount
needed (Taylor et al., 1990).
The lack of data collected from the isokinetic dynamometer to quantify for mus-
cle damage following the unaccustomed eccentric exercise (i.e. comparing set 1 to
set 6), for study 3, represents a potential limitation. Unable to account for the mag-
nitude of muscle damage between the experimental groups (low- and high-intensity
passive static stretching and control) prevented the assurance that all three groups
started from the same level of muscle damage. However, the choice to not take into
192 7 Limitations
account these data points was influenced by the description of the muscle damaging
protocol based on the study by Paschalis et al. (Paschalis et al., 2007).
Compliance
Limiting non-compliance amongst participants is essential for the proper execution

and completion of randomised controlled and clinical trials (Oba, Morita, Rahman,
& Sakamoto, 2006). Although measures were taken to promote and maximise par-
ticipant compliance with use of verbal and written instruction(s), non-compliance
represents a potential limitation. In study 3, unlike studies 1 and 2, where partici-
pants were under constant supervision, participants for the stretching groups were
instructed to perform the stretches at home. During the muscle function tests (eccen-
tric and isometric peak torque), although participants were encouraged verbally, the
discrepancy in baseline values represents a compliant issue, since participants for
the low-intensity passive static stretching group performed better. The inability to
control for activities outside the laboratory setting may also account for this discrep-
ancy. Non-compliance is an inherent issue with research studies, that can signifi-
cantly influence the outcome of the intervention(s), compromising an investigation
(Whitney & Dworkin, 1997). Low levels of compliance results in a lack of or reduc-
tion in power in detecting the effects of the intervention, which is potentially respon-
sible for the reporting on false negatives, leading to the rejection of effective
interventions (Campbell et al., 2000; Moher, Jones, & Lepage, 2001; Moher, Schulz,
& Altman, 2001). False negatives refer to a result that fails to detect a true difference
in the intervention or the effect (Brookes et al., 2001). In addition, less compliancy
from participants reduces the external validity of a study, in other words, how gen-
eralisable can the results be from the study (Kehoe, Chheda, Sahariah, Baird, &
Fall, 2009).
Nutrition
Another limitation is the lack of controlling the nutritional intake prior to all three
studies, since participants were not given a list of foods to avoid post intervention,
and for the duration of the studies. According to Esposito et al., an intake of macro-
nutrients produces oxidative stress and inflammatory responses (Esposito &
Giugliano, 2006). A macronutrient refers to nutrients required in large amounts
that provides the necessary energy to carry out daily activities, as well as maintain-
ing body functions (Nz, 2011). For instance, ingestion of glucose is associated with
an increase in leukocytes and mononuclear cells, and in the amount of nuclear factor
(NF)-κΒ (Dandona, Aljada, Chaudhuri, Mohanty, & Garg, 2005). Interestingly,
NF-κΒ has been associated with regulating the activity of at least 125 genes, most
of which are pro-inflammatory (Dandona et al., 2005).
Summary 193
Mental Stress
Although participants willingly allowed for the collection of blood (studies 1 and
2), some participants exhibited anxiety with the insertion of the vacutainer needle
into their veins. Depending on the nature of the stressor, evidence exists that psy-
chological stress can suppress or enhance immune functions (Kunz-Ebrecht,
Mohamed-Ali, Feldman, Kirschbaum, & Steptoe, 2003; Maes et al., 1998), with
studies suggesting the production of pro-inflammatory cytokines (Kunz-Ebrecht
et al., 2003; Maes et al., 1998; Steptoe, Willemson, Owen, Flower, & Mohamed-
Ali, 2001). According to Shephard, the acute stress response is marked by an
increase in IL-1ra and IL-6 which may take several minutes to evoke (Shephard,
2002). The mental stress during the high-intensity passive static stretching for study
1 and 2 may have contributed to increased levels of the blood biomarkers. However,
with the nature and the design of these studies, it would have been very difficult to
control or eliminate for mental stress.
Age
All three studies made use of recreationally active young males (average age
26 years). Although they referred to the intensity of a stretch and were not con-
cerned with the increase or decrease in ROM, it would have been interesting to
observe if a difference existed in considering age. According to a study, changes
amongst the elderly include a decrease in muscle mass associated with fibre atrophy
and hyperplasia, and a remodelling of the motor unit (Feland, Myrer, & Merrill,
2001). The decline of skeletal muscle mass occurs at an average rate of 4% until
50 years of age, increasing to 10% per decade (Fielding, 1995). Therefore, it would
have been very interesting to observe how the magnitude and rate of stretching
intensity may affect the response of older muscle with regard to the measurement of
inflammatory biomarkers.
Summary
Despite the strengths of the three studies (i.e. randomisation, quantification of the
stretching intensities, one-on-one instruction), there were limitations. The use of
methods or tools to minimise non-compliance and mental stress, methodological
consistency, a greater age range (i.e. elderly patients), and the potential lack of suf-
ficient statistical power to detect differences between conditions for outcomes. The
unsupervised nature of the passive static stretching interventions (study 3), and the
lack of blinding of the assessor to the assessments of the studies, are potential limi-
tations as well. In addition, timing for collection of blood samples, the half-lives for
194 7 Limitations
the pro-inflammatory cytokines [(IL-1β, TNF-α, and IL-6), study 1], and the mea-
suring for IL-6 (study 2) would have provided a thorough indication of the effect of
the magnitude and the rate of stretching intensity relative to inflammation and the
inflammatory response(s).
References
Akira, S., Hirano, T., Taga, T., & Kishimoto, T. (1990). Biology of multifunctional cytokines: il-6
and related molecules (Il-1 and Tnf). FASEB, 4, 2860–2867.
Bauer, J., Lengyel, G., Bauer, T. M., Acs, G., & Gerok, W. (1989). Regulation of interleukin-6
receptor expression in human monocytes and hepatocytes. FEBS Letters, 249, 27–30.
Brookes, S. T., Whitley, E., Peters, T. J., Mulheran, P. A., Egger, M., & Davey Smith, G. (2001).
Subgroup analyses in randomised controlled trials: quantifying the risks of false-positives and
false-negatives. Health Technology Assessment, 5, 1–56.
Campbell, M., Fitzpatrick, R., Haines, A., Kinmouth, A. L., Sandercock, P., & Spiegelhalter, D.
(2000). Framework for design and evaluation of complex interventions to improve health. BMJ,
32, 694–696.
Dandona, P., Aljada, A., Chaudhuri, A., Mohanty, P., & Garg, R. (2005). A comprehensive per-
spective based on interactions between obesity, diabetes, and inflammation. Circulation, 111,
1448–1454.
Esposito, K., & Giugliano, D. (2006). Diet and inflammation: a link to metabolic and cardiovascu-
lar disease. European Heart Journal, 27, 15–20.
Feland, J. B., Myrer, J. W., & Merrill, R. M. (2001). Acute changes in hamstring flexibility: pnf
versus static stretch in senior athletes. Physical Therapy in Sport, 2, 186–193.
Fielding, R. A. (1995). Symposium on ‘nutrition for the elderly’. The Proceedings of the Nutrition
Society, 54, 665–675.
Fuller, G. M., & Grenett, H. E. (1989). The structure and function of the mouse hepatocyte stimu-
lating factor. Annals of the New York Academy of Sciences, 557, 31–44.
Gabay, C., & Kushner, I. (1999). Acute-phase proteins and other systemic responses to inflamma-
tion. The New England Journal of Medicine, 340, 448–454.
Hazuda, D. J., Lee, J. C., & Young, P. R. (1988). The kinetics of interleukin 1 secretion from acti-
vated monocytes. The Journal of Biological Chemistry, 263, 8473–8479.
Kehoe, S. H., Chheda, P. S., Sahariah, S. A., Baird, J., & Fall, C. H. D. (2009). Reporting of par-
ticipant compliance in randomized controlled trials of nutrition supplements during pregnancy.
Maternal & Child Nutrition, 5, 97–103.
339–342.
Kunz-Ebrecht, S. R., Mohamed-Ali, V., Feldman, P. J., Kirschbaum, C., & Steptoe, A. (2003).
Cortisol responses to mild psychological stress are inversely associated with proinflammatory
cytokines. Brain, Behavior, and Immunity, 17, 373–383.
Lacy, P., & Stow, J. L. (2011). Cytokine release from innate immune cells: association with diverse
membrane trafficking pathways. Blood, 118, 9–18.
Maes, M., Song, C., Lin, A., De Jongh, R., Van Gastel, A., Kenis, G., et al. (1998). The effects of
psychological stress on humans: increased production of pro-inflammatory cytokines and a
th1-like response in stress induced anxiety. Cytokine, 10, 313–318.
References 195
Mcfarland-Mancini, M. M., Funk, H. M., Paluch, A. M., Zhou, M., Giridhar, P. V., Mercer, C. A.,
et al. (2010). Differences in wound healing in mice with deficiency of Il-6 versus Il-6 receptor.
Journal of Immunology, 184, 7219–7228.
Moher, D., Jones, A., & Lepage, L. (2001a). Use of the consort statement and quality of reports of
randomized trials: a comparative before-and-after evaluation. JAMA, 285, 1992–1995.
Moher, D., Schulz, K. F., & Altman, D. (2001b). The consort statement: revised recommendations
for improving quality of reports of parallel-group randomized trials. JAMA, 285, 1987–1991.
Nz, R. (2011). Macronutrients. New York: Plenum Press.
Oba, K., Morita, S., Rahman, M., & Sakamoto, J. (2006). Factors associated with compliance and
non-compliance by physicians in a large-scale randomized clinical trial. Trials, 7, 1–7.
Oliver, J. C., Bland, L. A., Oettinger, C. W., Arduino, M. J., Mcallister, S. K., Aquero, S. M., et al.
(1993). Cytokine kinetics in an in vitro whole blood model following an endotoxin challenge.
Lymphokine and Cytokine Research, 12, 115–120.
Paschalis, V., Giakas, G., Baltzopoulos, V., Jamurtas, A. Z., Theoharis, V., Kotzamanidis, C., et al.
(2007). The effects of muscle damage following eccentric exercise on gait biomechanics. Gait
& Posture, 25, 236–242.
Pedersen, B. K. (2000). Exercise and cytokines. Immunology and Cell Biology, 78, 532–535.
Rohde, T., Maclean, D. A., Richter, E. A., Kiens, B., & Pedersen, B. K. (1997). Prolonged sub-
maximal eccentric exercise is associated with increased levels of plasma Il-6. The American
Journal of Physiology, 273, E85–E91.
Samols, D., Agrawal, A., & Kushner, I. (2002). Acute phase proteins. Cytokine Reference On-line,
(Feldman, J.J. and Oppenheim, J.J., ed). Academic Press, Harcourt, London, 1–16.
Shephard, R. J. (2002). Cytokine responses to physical activity, with particular reference to Il-6:
sources, actions, and clinical implications. Critical Reviews in Immunology, 22, 165–182.
Steptoe, A., Willemson, G., Owen, N., Flower, L., & Mohamed-Ali, V. (2001). Acute mental stress
elicits delayed increases in circulating inflammatory cytokine levels. Clinical Science, 101,
185–192.
France), 47, 661–673.
muscle-tendon units. The biomechanical effects of stretching. The American Journal of Sports
Medicine, 18, 300–309.
Whitney, C. W., & Dworkin, S. F. (1997). Practical implications of noncompliance in randomized
clinical trials for temporomandibular disorders. Journal of Orofacial Pain, 11, 130–138.
Chapter 8
Future Research
Introduction
Studies investigating low-intensity stretching (i.e. microStretching) compared to

static stretching (Wyon, Felton, & Galloway, 2009), as well as moderate- and high-
intensity stretching (Wyon, Smith, & Koutedakis, 2013), observed that both active
and passive ranges of motion improved with low-intensity stretching. Although,
passive and active range of motion was not measured within these studies, simi-
larly to the aforementioned studies, low-intensity passive static stretching was
observed to be more beneficial compared to high-intensity passive static stretching
and no stretching. With study one high-intensity passive static stretching was asso-
ciated with inflammation (i.e. increase in hsCRP levels), with study two reporting
that both low- (30%) and moderate (60%)-intensity passive static stretching was
not associated with inflammation, confirmed by effect sizes. In study three,
RMANOVA and the magnitude-based inference approach suggested that low- ver-
sus high-intensity passive static stretching and no stretching (control) was linked to
faster recovery times, measured by a decrease in perceived muscle soreness and an
increase in muscle function (eccentric and isometric peak torque). In Chap. 2,
mechanotransduction was introduced, conceivably suggesting that it may be the
mechanism of how the magnitude of stretching (mechanical force) may be respon-
sible for a biochemical response (inflammation). Calpains, an activated “calcium-
dependent protease”, expressed because of the body’s inability to buffer the release
of Ca2+, was proposed as the interface between the mechanical disruption and the
biochemical response. The attenuation or delay of desmin disruption and Z-disc
streaming with the use of a calcium channel blocker (Beaton, Tarnopolsky, &
Phillips, 2002) illustrates the importance of loss of calcium homeostasis to the
plausible yet reasonable theory of stretching intensity on the onset or mitigation of
the inflammatory response. Further research, specifically focused on mechano-
transduction, calpains, inflammation (blood biomarkers), and stretching intensity
(low, moderate, or high), will provide an understanding of how this mechanical

198 8 Future Research
force (stretching) influences the hierarchy of structures from the macro (muscle,
tendons, MTU) to the micro (cells, ECM), and the recovery of muscle. Such an
awareness will provide coaches and medical practitioners (physicians, therapists,
etc.) with an insight to the effects and outcomes associated with low- and high-
intensity passive static stretching, allowing for the better design of training, reha-
bilitation, and recovery programmes. For instance, if inflammation is already
present in connective and soft tissue of the musculoskeletal system (i.e. rheuma-
toid- and osteoarthritis), investigating whether low-intensity passive static stretch-
ing disrupts or attenuates the inflammatory response may provide an adjunct
intervention to medication. In addition, since low-intensity passive static stretching
is not associated with pain and discomfort, with pain affiliated with mental/emo-
tional stress (Gatchel, 2004; Melzack & Katz, 1999; Merskey & Bogduk, 1994), it
is plausible that low-intensity passive static stretching may provide relief from this
stress. Similar to physical stress, the release of pro-inflammatory cytokines (IL-1,
TNF-α, and TNF-α) has been identified with mental/emotional stress, suggesting
their involvement in pathways influencing the psychosocial health of individuals
(Steptoe, Willemson, Owen, Flower, & Mohamed-Ali, 2001).
Further Research
Further Research Based on Studies
Subject to the results of the three studies, further research is justified, investigating
the effects of stretching intensity on acute and chronic inflammation and recovery
of the muscle from injury, trauma, and damage. These investigations will benefit
from the recruitment of a greater population of participants. Although power calcu-
lations for study one and two indicated that 11 participants equates to 80% power,
based on the primary outcome (hsCRP), unfortunately, missing data for hsCRP for
three participants in study one resulted in the study being underpowered (n = 9).
An improved design with regard to blood collection (i.e. half-lives) for the pro-
inflammatory cytokines (IL-1β, IL-6, and TNF-α) will provide a better understand-
ing of how the magnitude of stretching intensity (mechanical stimulus) influences
inflammation (biochemical response). These pro-inflammatory cytokines form a
collaborative network, with expression of each influencing the other. For instance,
IL-1β and TNF-α are both potent induces of IL-6, with IL-6 regulating the expres-
sion of TNF-α (Akira, Hirano, Taga, & Kishimoto, 1990; Akira, Taga, & Kishimoto,
1993; McGee, Bamberg, Vitukus, & McGhee, 1995). This synergistic relationship
is an attempt to regulate the immune response and the inflammatory reaction (Akira
et al., 1990, 1993; Streetz, Wustefeld, Klein, Manns, & Trautwein, 2001). However,
with IL-6 acting as the primary mediator of the acute phase response, and the syn-
thesis of C-reactive protein (Gruys, Toussaint, Niewold, & Koopmans, 2005;
Ramadori, Van Damme, Rieder, & Meyer Zum Buschenfelde, 1988; Streetz et al.,
2001), this cytokine with hsCRP would be a primary choice for future studies, since
Further Research 199
IL-1β and TNF-α are not implicated as primary activators of an acute phase response
(Akira et al., 1990). Blood collection will coincide with the half-lives for IL-6
(i.e. 2–4 h) (Pedersen & Hoffman-Goetz, 2000) and hsCRP (19–20 h) (Marino &
Giotta, 2008; Pepys & Hirschfield, 2003). In addition, since a positive correlation
was observed between IL-6 and creatine kinase (Bruunsgaard et al., 1997), and with
creatine kinase used as a marker of muscle damage related to both the intensity and
duration of exercise (Noakes, 1987), future research needs to measure for plasma
creatine kinase.
In designing the three studies, the intention was to provide a logical progression
investigating the influence of the magnitude of stretching. The effect of high-
intensity passive static stretching (study one) was compared to both low- and
moderate-intensity passive static stretching (study two), in an attempt to determine
how these different magnitudes of stretching influence the acute inflammatory
response, as well as perceived muscle soreness and muscle function (study three).
Results from the first two studies revealed that high-intensity passive static stretch-
ing was associated with an increase in hsCRP, compared to low- and moderate-
intensity passive static stretching, and that low-intensity passive static stretching
was beneficial for aiding the recovery of muscle from unaccustomed eccentric exer-
cise (study three). However, flaws with the methodology of the three studies need to
be addressed with future studies. The high-intensity stretching levels were different
(80% study one, 90% study two, and a range for study three, 70–80%), with the
low-intensity levels for studies two and three being different as well (30% for study
two and a range for study three, 30–40%). The frequency of stretching was also dif-
ferent, with participants for studies one and three, performing three sets, and five
sets for study two. Although evidence in the literature suggests that five sets elicits
a similar response to three sets, with no statistical difference existing (Taylor,
Dalton, Seaber, & Garrett, 1990), future studies will look to address this inconsis-
tency. Besides addressing the issues of blood collection, participant size, and the
inconsistencies of stretching intensity and frequency, based on the results obtained,
the sections below provide suggestions for future research projects to address the
influence of the magnitude of stretching intensity and the inflammatory response.
Ultrasonography
This non-invasive technique has been used to determine the stiffness and hysteresis,
elastic characteristics, and the viscoelastic properties of the human tendon struc-
tures in vivo in real time (Fukashiro, Itoh, Ichinose, Kawakami, & Fukunaga, 1995;
Kubo, Kanehisa, & Fukunaga, 2001, 2002). In the past, research investigating the
effect of stretching on the properties of both connective and soft tissue were limited
to animal studies (Stromberg & Wiederhielm, 1969; Taylor et al., 1990; Viidik,
1972), often questioning the applicability of the results to humans because of the
differences existing amongst species (Ker, Alexander, & Bennett, 1988). With ultra-
sonography, one observes the response of the tissues to the mechanical stress/strain
within their natural environment, providing real-time data of the tendon and
aponeuroses, allowing for a better understanding of the structure and function of the
tendinous tissues (Muramatsu et al., 2001). To investigate the response of low- and
high-intensity passive static stretching on connective and soft tissue, compression
(strain) ultrasound elastography would be employed. This method, which assesses
the mechanical properties of tissue through the application of stress, allows for
detection of tissue displacement and the construct of an image (Drakonaki & Allen,
2012). A correlation between quantitative strain ultrasound elastography parameters
and elevated serum markers suggests its importance in staging and monitoring of
inflammation (Botar-Jid et al., 2010). Observing the response of the connective tis-
sue and muscle fibres in real time may provide answers as to why low-intensity
passive static stretching was associated with a better muscle function and recovery
from soreness.
Reactive Oxygen Species
As alluded to above, tissue damage is associated with the activation of the acute
inflammatory response responsible for the secretion of various cytokines and che-
mokines in order to recruit immune cells [i.e. neutrophils and macrophages (pro-
inflammatory)] to the site of injury. Both studies one and two observed an increase
in levels of hsCRP, a marker of inflammation, in response to high-intensity passive
static stretching. According to Tidball (2005), inflammatory cells can promote both
injury and repair through the combined actions of free radicals, growth factors, and
chemokines. Neutrophils, the first cells to adhere to the vascular wall, migrate
across the wall at the site of injury, activating the process of phagocytosis, as well
as secreting vasoactive and pro-inflammatory mediators (cytokines, chemokines,
extracellular matrix components) (Clark & Kupper, 2005; Kolaczkowska & Kubes,
2013), promoting the release and activation of reactive oxygen species (Closa &
Folch-Puy, 2004; Farber, 1994). These species, partially reduced metabolites of
oxygen, possess a hormetic quality, a biphasic nature, describing the response of the
cell or organism to a low-dose stimulation (beneficial) or high-dose inhibitory
(toxic) effect to an environmental agent (Mattson, 2008). The local persistence of
reactive oxygen species, sustained by infiltrated neutrophils, defines a high-dose
toxic effect, responsible for further damage, by oxidatively damaging differentiat-
ing myoblasts and myotubes, thereby delaying the restoration of muscle to its origi-
nal condition (Barbieri & Sestili, 2012). However, in combination with growth
factors and chemokines, reactive oxygen species participate in a cascade of events
responsible for the regeneration and repair of muscle (Barbieri & Sestili, 2012;
Droge, 2002), playing an important role in skeletal muscle function and adaptation
to exercise (Powers, Ji, Kavazis, & Jackson, 2011). The extent to which the inflam-
matory cells promote damage is determined by the acute use of the muscle during
the time of damage and the intensity and severity of the exercise (Tidball, 2005).
Therefore, it would be very interesting to investigate whether stretching intensity

(high or low) influences the release and concentration of the neutrophils and
macrophages responsible for oxidative stress in humans, since passive stretching
was associated with the release of neutrophils in adult male mice (Pizza, Koh,
McGregor, & Brooks, 2002).
Musculoskeletal Disorders and Pain
Musculoskeletal pain accounts for between 13.5 and 47% of the general population,
with a huge burden projected on the public healthcare system (Cimino, Ferrone, &
Cutolo, 2011). Within the clinical setting, future research could investigate the influ-
ence of low-intensity passive static stretching on musculoskeletal disorders (i.e.
osteo- and rheumatoid arthritis and low back pain). In study three, we observed both
a decrease in perceived muscle soreness and an increase in muscle function, sug-
gesting that low-intensity passive static stretching may offer relief from pain, as
well as aid in the recovery of connective tissue and muscle from damage associated
with these disorders.
Musculoskeletal disorders, either acute or chronic, consist of conditions where
part of the system is injured or affected over time, with symptoms including pain,
dysfunction, and discomfort in the bones, joints, muscles, or surrounding structures
(Verhagen, Cardoso, & Bierma-Zeinstra, 2012). The aetiology is related to a variety
of activities or causes such as sports, work, fractures, contusions, degenerative
(osteoarthritis), or systemic (rheumatoid arthritis) diseases, as well as increased
ageing (Felson, 2000; Verhagen et al., 2012).
Pain and loss of function are common to musculoskeletal disorders (Atzeni
et al., 2011; Cimino et al., 2011; Verhagen et al., 2012), with the international asso-
ciation for the study of pain, defining pain as an unpleasant sensory and emotional
experience associated with actual or potential tissue damage or described in terms
of such damage (Crombie, Croft, Linton, Lereasche, & Von Koff, 1999). In the
clinical setting, it is an index for the severity and activity of the underlying condi-
tion, as well as a prognostic/therapeutic indicator, defined by the use of health
resources (Cimino et al., 2011). Presently, exercises aimed at reducing pain associ-
ated with musculoskeletal disorders include both land (walking, resistance, stretch-
ing) and aquatic (water-based exercises, aquatic therapy or hydrotherapy) (Silva
et al., 2008; Wang, Belza, Thompson, Whitney, & Bennett, 2001), with the design
and prescription of these exercises adhering to the parameters of training and
stretching: intensity (magnitude), duration, and frequency (rate) (Marschall, 1999;
Mujika et al., 1995). Therefore, the complaint of pain and swelling in joints associ-
ated with several musculoskeletal disorders (i.e. osteo- and rheumatoid arthritis)
may determine the intensity, duration, frequency, and adherence of individuals to
exercise.
Recovery and Regeneration
With athletes training harder and longer than ever before, recovery and regeneration
is very important, since increases in physical loads exceeding physiological norms
are responsible for changes in the athlete’s body, lowering their capacity to maxi-
mise training and competition (Apostolopoulos, 2004, 2010). The athlete’s ability
to tolerate load depends on recovery, for a rapid rate of recovery suggests that the
training load can be advanced (Apostolopoulos, 2004, 2010). Maximising athletic
performance is a balance between the optimal amount of physical training and
proper recovery periods, allowing for the greatest adaptation from competition and
training (Coutts & Aoki, 2009; Gamble, 2006). To achieve this, coaches need to
develop physical and sport-specific conditioning programmes aimed at improving
performance (anabolic process—adaptation) while minimising damage and injury
(catabolic process—maladaptation) (Apostolopoulos, 2004, 2010; Berdej-del-
Fresno & Laupheimer, 2014; Gamble, 2006; Kentta & Hassmen, 1998). As previ-
ously discussed, muscle damage results in disruption in the plasma-lemma of the
tissue (Jarvinen & Lehto, 1993; Kalimo & Jarvinen, 1997), activating an
extracellular-regulated protein kinase cascade initiating local inflammation
(Aronson et al., 1998; Li, Cummins, & Huard, 2001). Since muscle damage from
training is part of professional and recreational sports (Li et al., 2001) affecting the
body’s homeostasis directly (muscle lacerations and contusions) or indirectly (isch-
emia, excessive stress or strain, and neurological dysfunction) (Garrett, Safran,
Seaber, Glisson, & Ribbeck, 1987; Kasemkijwattana et al., 1998), recovery and
regeneration are the processes for restoring homeostasis. This restoration enables
the body to adapt its functions and system to a higher level (Koutedakis, Metsios, &
Stavropoulos-Kalinoglou, 2006).
To understand the processes of adaptation to load, one must consider the contri-
bution of the physiological, psychological, biochemical, and immunological fac-
tors, both independently and in combination (Kentta & Hassmen, 1998). These
factors influence and are influenced by the simultaneous increase in antagonistic
mediators of exercise, the anabolic (i.e. growth hormone, insulin-like growth factor)
(Adams, 2002; Schwarz, Brasel, Hintz, Mohan, & Cooper, 1996) and catabolic (i.e.
pro-inflammatory cytokine—IL-6) (Nemet, Oh, Kim, Hill, & Cooper, 2002;
Ostrowski, Rohde, Asp, Schjerling, & Pedersen, 1999). Therefore, disruption of the
integrity of the muscle tissue and cells is a balance between the anabolic and cata-
bolic mediators and the four factors mentioned above, collectively responsible for
proper recovery and regeneration.
For an athlete to improve their performance, the timing for recovery and regen-
eration is vital, facilitating an anabolic response. A prime example is DOMS, with
the intensity of discomfort increasing within the first 24 h post exercise, peaking
between 24 and 72 h (Armstrong, 1984; Miles & Clarkson, 1994; Talag, 1973). As
mentioned above, DOMS is associated with negative consequences, responsible for
temporarily restricting the ability to perform activity (Clarkson & Hubal, 2002;
Clarkson, Nosaka, & Braun, 1992; Newham, Mills, Quigley, & Edwards, 1983;
Vasudevan, 1993). In addition, according to Smith (1991), the underlying

mechanism for DOMS is acute inflammation with the sensation of soreness repre-
senting inflammatory pain (Lieber & Friden, 1993; Macintyre, Reid, & McKenzie,
1995; Smith, 1991), influenced by the extent of, and response to, muscle damage
(Tidball, 2005). Both Faulkner, Brooks, & Opiteck (1993) and Warren, Lowe, &
Armstrong (1999) suggest that prolonged strength loss is indirectly the most valid
way to quantitatively assess for changes in the magnitude of muscle damage with
time. Therefore, actions alleviating and hastening recovery (i.e. diminished magni-
tude of the inflammatory response) would enhance restoration of maximal function
of muscle (Cheung, Hume, & Maxwell, 2003). With low-intensity passive static
stretching associated with a decrease in perceived muscle soreness, and an increase
in muscle function (eccentric and isometric peak torque) (study three), with no
inflammation (study two), a future study would investigate its effects on the recov-
ery and regeneration of the musculoskeletal system from various bouts of training
load and intensity (low, medium, high), specifically, whether the magnitude and rate
of stretching expedites the healing process of the damaged tissue, allowing for a
better adaptation of the body to a training load. Parallel to this study, another inves-
tigation would consider whether stretching intensity (low, medium, and high) is
either an anabolic or catabolic catalyst, since currently no therapy exists producing
complete regeneration and 100% full functional recovery (Li et al., 2001).
Relaxation Response
With the stretching groups performing their respective stretching exercises prior to
sleep (study 3), an interesting response voiced by the low-intensity passive static
stretching group was the quality of their sleep (deeper). Unlike the high-intensity
passive static group, which stretched to the point of discomfort with slight pain, this
group performed stretches similar to a warm gentle feeling. Although the informa-
tion conveyed was anecdotal, it prompts the need to investigate whether low-
intensity passive static stretching may promote a relaxation response.
The relaxation response refers to a hypothesised integrated feedback that results
in the generalised decrease in activity of the sympathetic nervous system (SNS)
(Beary & Benson, 1974), the fight-or-flight response, both physiologically and psy-
chologically (Bhasin et al., 2013). It is a physical state of deep rest, changing physi-
cal and emotional responses to stress, as well as being associated with a decrease in
muscle tone (Benson, 2000). It has been observed that the activation of the SNS
facilitates inflammation through the induction of C-reactive protein and pro-
inflammatory cytokine (IL-1, IL-6, and TNF-α) production (Elenkov, Iezzoni, Daly,
Harris, & Chrousos, 2005; Grebe et al., 2010), with evidence suggesting that these
inflammatory mediators factor in the pathogenesis of major depression, metabolic
syndromes, and sleep disturbances (Elenkov et al., 2005). Sleep has a restorative
role (Vyazovskiy & Harris, 2013), providing opportunities for processes such as the
synthesis of macromolecules (Mackiewicz et al., 2007), recuperation from oxidative
stress or toxins accumulated during wakefulness (Inoue, Honda, & Komoda, 1995;
Reimund, 1994), and the replenishment of energy stores such as glycogen
(Benington & Heller, 1995; Scharf, Naidoo, Zimmerman, & Pack, 2008). Therefore,
it would be very interesting to investigate if a relaxation response occurs with the
use of low-intensity passive static stretching and, if so, how this influences the qual-
ity of sleep, and the subsequent recovery from training, muscle damage, and injury.
References
Adams, G. R. (2002). Autocrine/paracrine IGF-I and skeletal muscle adaptation. Journal of
Apostolopoulos, N. (2004). Microstretching-a new recovery and regeneration technique. New
Studies in Athletics, 19, 47–54.
Apostolopoulos, N. (2010). microStretching-A practical approach for recovery and regeneration.
Armstrong, R. B. (1984). Mechanisms of exercise-induced delayed onset muscular soreness: A
brief review. Medicine and Science in Sports and Exercise, 16, 529–538.
Aronson, D., Wojtaszewski, J. F., Thorell, A., Nygren, J., Zangen, D., Richter, E. A., et al. (1998).
Extracellular-regulated protein kinase cascades are activated in response to injury in human
skeletal muscle. American Journal of Physiology. Cell Physiology, 275, C555–C561.
Atzeni, F., Cazzola, M., Benucci, M., Di Franco, M., Salaffi, F., & Sarzi-Puttini, P. (2011). Chronic
widespread pain in the spectrum of rheumatological diseases. Best Practice & Research:
Clinical Rheumatology, 25, 165–171.
Barbieri, E., & Sestili, P. (2012). Reactive oxygen species in skeletal muscle signaling. Journal of
Signal Transduction, 2012, 1–17.
Beary, J. F., & Benson, H. (1974). A simple psychophysiologic technique which elicits the hypo-
metabolic changes of the relaxation response. Psychosomatic Medicine, 36, 115–120.
Beaton, L. J., Tarnopolsky, M. A., & Phillips, S. M. (2002). Contraction-induced muscle damage
in humans following calcium channel blocker administration. The Journal of Physiology, 544,
849–859.
Benington, J. H., & Heller, H. C. (1995). Restoration of brain energy metabolism as a function of
sleep. Progress in Neurobiology, 45, 347–360.
Benson, H. (2000). The relaxation response. New York: Harper Collins.
Berdej-del-Fresno, D., & Laupheimer, M. W. (2014). Recovery and regeneration behaviours in
elite English futsal players. Am. Journal of Sports Science and Medicine, 2, 77–82.
Bhasin, M. K., Dusek, J. A., Chang, B.-H., Joseph, M. G., Denninger, J. W., Fricchione, G. L.,
et al. (2013). Relaxation response induces temporal transcriptome changes in energy metabo-
lism, insulin secretion and inflammatory pathways. PLoS One, 8, 1–13.
Botar-Jid, C., Damian, L., Dudea, S. M., Vasilescu, D., Rednic, S., & Badea, R. (2010). The
contribuition of ultrasonography and sonoelastography in assessment of myositis. Medical
Ultrasonography, 12, 120–126.
Bruunsgaard, H., Galbo, H., Halkjaer, K. J., Johansen, T. L., Maclean, D. A., & Pedersen, B. K.
(1997). Exercise-induced increase in serum interleukin-6 in humans is related to muscle dam-
age. The Journal of Physiology, 499, 833–841.
References 205
Cimino, M. A., Ferrone, C., & Cutolo, M. (2011). Epidemiology of chronic musculoskeletal pain.
Best Practice & Research: Clinical Rheumatology, 25, 173–183.
Clark, R., & Kupper, T. (2005). Old meets new: The interaction between innate and adaptive
immunity. The Journal of Investigative Dermatology, 125, 629–637.
Clarkson, P. M., & Hubal, M. J. (2002). Exercise-induced muscle damage in humans. American
journal of physical medicine & rehabilitation, 81, S52–S69.
Clarkson, P. M., Nosaka, K., & Braun, B. (1992). Muscle function after exercise-induced muscle
damage and rapid adaptation. Medicine and Science in Sports and Exercise, 24, 512–520.
Closa, D., & Folch-Puy, E. (2004). Oxygen free radicals and the systemic inflammatory response.
IUBMB Life, 56, 185–191.
Coutts, A. J., & Aoki, M. S. (2009). Monitoring training in team sports. Olympic Laboratory:
Technical Scientific Bulletin of the Brazilian Olympic Committee, 9, 1–3.
Crombie, I. K., Croft, P., Linton, S. J., Lereasche, L., & Von Koff, M. (1999). Epidemiology of
pain. Seattle, WA: IASP Press.
Drakonaki, E. E., & Allen, G. M. (2012). Ultrasound elastography for musculoskeletal applica-
tions. The British Journal of Radiology, 85, 1435–1445.
Droge, W. (2002). Free radicals in the physiological control of cell function. Physiological
Reviews, 82, 47–95.
Elenkov, I. J., Iezzoni, D. G., Daly, A., Harris, A. G., & Chrousos, G. P. (2005). Cytokine dysregu-
lation, inflammation and well being. Neuroimmunomodulation, 12, 255–269.
Farber, J. L. (1994). Mechanisms of cell injury by activated oxygen species. Environmental Health
Perspectives, 102, 17–24.
contractions: Conditions of occurrence and prevention. Physical Therapy, 73, 911–921.
Felson, D. T. (2000). Epidemiology of the rheumatic diseases. Philadelphia: Lippincott, Williams
& Collins.
Fukashiro, S., Itoh, M., Ichinose, Y., Kawakami, Y., & Fukunaga, T. (1995). Ultrasonography gives
directly but noninvasively elastic characteristic of human tendon in vivo. European Journal of
Gamble, P. (2006). Periodization of training for team sports athletes. Strength and Conditioning
Journal, 28, 56–66.
Garrett, W. E., Safran, M., Seaber, V. A., Glisson, R. R., & Ribbeck, M. B. (1987). Biochemical
comparison of simulated and non-simulated skeletal muscle pulled to failure. The American
Gatchel, R. J. (2004). Comorbidity of chronic pain and mental health disorders: The biopsychoso-
cial perspective. The American Psychologist, 59, 795–805.
Grebe, K. M., Takeda, K., Hickman, H. D., Bailey, A. L., Embry, A. C., Bennick, J. R., et al.
(2010). Cutting edge: Sympathetic nervous system increases proinflammatory cytokines and
exacerbates influenza a virus pathogenesis. Journal of Immunology, 184, 540–544.
Inoue, S., Honda, K., & Komoda, Y. (1995). Sleep as neuronal detoxification and restitution.
Behavioural Brain Research, 69, 91–96.
Jarvinen, M. J., & Lehto, M. U. K. (1993). The effects of early mobilisation and immobilisation on
the healing process following muscle injuries. Sports Medicine, 15, 78–89.
Kalimo, H., & Jarvinen, M. J. (1997). Muscle injuries in sports. Bailliere’s Clinical Orthopaedics,
2, 1–24.
Kasemkijwattana, C., Menetrey, J., Dai, C. S., Bosch, P., Buranapanitkit, B., Moreland, M. S.,
et al. (1998). Biologic intervention in muscle healing and regeneration. Sports Medicine and
Arthroscopy Review, 6, 95–102.
Kentta, G., & Hassmen, P. (1998). Overtraining and recovery. A conceptual model. Sports
Medicine, 26, 1–16.
Ker, R. F., Alexander, R. M., & Bennett, M. B. (1988). Why are mammalian tendons so thick?
Journal of Zoology, London, 216, 309–324.
Kolaczkowska, E., & Kubes, P. (2013). Neutrophil recruitment and function in health and inflam-
mation. Nature Reviews Immunology, 13, 159–175.
Koutedakis, Y., Metsios, G. S., & Stavropoulos-Kalinoglou, A. (Eds.). (2006). Periodization of
exercise training in sport. Philidelphia: Churchill-Livingstone Elsevier.
Kubo, K., Kanehisa, H., & Fukunaga, T. (2001). Effect of stretching training on the viscoelastic
propertties of human tendon structures in vivo (abstract). Journal of Ultrasound in Medicine,
20, 128.
Kubo, K., Kanehisa, H., & Fukunaga, T. (2002). Effect of stretching training on the viscoelastic
properties of human tendon structures in vivo. Journal of Applied Physiology, 92, 595–601.
Macintyre, D. L., Reid, W. D., & McKenzie, D. C. (1995). The inflammatory response to muscle
Mackiewicz, M., Shockley, K. R., Romer, M. A., Galante, R. J., Zimmerman, J. E., Naidoo, N.,
et al. (2007). Macromolecular biosynthesis: A key function of sleep. Physiological Genomics,
31, 441–457.
Mattson, M. P. (2008). Hormesis defined. Ageing Research Reviews, 7, 1–7.
McGee, D. W., Bamberg, T., Vitukus, S. J. D., & McGhee, J. R. (1995). A synergistic relationship
between TNF-α, IL-1β, and TGF-β1 on IL-6 secretion by the IEC-6 intestinal epithelial cell
line. Immunology, 86, 6–11.
Melzack, R., & Katz, J. (Eds.). (1999). Pain measurements in persons with pain. London, UK:
Chruchill Livingstone.
Miles, M. P., & Clarkson, P. M. (1994). Exercise-induced muscle pain, soreness, and cramps. The
Journal of Sports Medicine and Physical Fitness, 34, 203–216.
20, 395–406.
Muramatsu, T., Muraoka, T., Takeshita, D., Kawakami, Y., Hirano, Y., & Fukunaga, T. (2001).
Mechanical properties of tendon and aponeurosis of human gastrocnemius muscle in vivo.
Journal of Applied Physiology, 90, 1671–1678.
Nemet, D., Oh, Y., Kim, H. S., Hill, M. A., & Cooper, D. M. (2002). The effect of intense exer-
cise onj inflammatory cytokines and growth mediators in adolescent boys. Pediatrics, 110,
681–689.
Newham, D. J., Mills, K. R., Quigley, B. M., & Edwards, R. H. T. (1983). Pain and fatigue after
concentric and eccentric contractions. Clinical Science, 64, 55–62.
4, 245–267.
Ostrowski, K., Rohde, T., Asp, S., Schjerling, P., & Pedersen, B. K. (1999). Pro- and anti-
inflammatory cytokine balance in strenuous exercise in humans. The Journal of Physiology,
515, 287–291.
References 207
Pedersen, B. K., & Hoffman-Goetz, L. (2000). Exercise and the immune system: Regulation,
integration and adaption. Physiological Reviews, 80, 1055–1081.
Pepys, M. B., & Hirschfield, G. M. (2003). C-reactive protein: a critical update. The Journal of
Pizza, F. X., Koh, T. J., McGregor, S. J., & Brooks, S. V. (2002). Muscle inflammatory cells after
Physiology, 92, 1873–1878.
Powers, S. K., Ji, L. L., Kavazis, A. N., & Jackson, M. J. (2011). Reactive oxygen species: Impact
on skeletal muscle. Comprehensive Physiology, 1, 941–969.
Reimund, E. (1994). The free radical flux theory. Medical Hypotheses, 43, 231–232.
Scharf, M. T., Naidoo, N., Zimmerman, J. E., & Pack, A. I. (2008). The energy hypothesis of sleep.
Progress in Neurobiology, 86, 264–280.
Schwarz, A. J., Brasel, J. A., Hintz, R. L., Mohan, S., & Cooper, D. M. (1996). Acute effect of
brief low- and high-intensity exercise on circulating IGF-I, II, and IGF binding protein-3 and
its proteolysis in young heathy men. The Journal of Clinical Endocrinology and Metabolism,
81, 3492–3497.
Silva, L. E., Valim, V., Pessanha, A. P. C., Myamoto, S., Jones, A., & Natour, J. (2008). Hydrotherapy
versus conventional land-based exercise for the management of patients with osteoarthritis of
the knee: A randomized clinical trial. Physical Therapy, 88, 12–21.
Steptoe, A., Willemson, G., Owen, N., Flower, L., & Mohamed-Ali, V. (2001). Acute mental stress
elicits delayed increases in circulating inflammatory cytokine levels. Clinical Science, 101,
185–192.
France), 47, 661–673.
Stromberg, D. D., & Wiederhielm, C. A. (1969). Viscoelastic description of a collagenase tissue in
simple elongation. Journal of Applied Physiology, 26, 857–862.
Medicine, 18, 300–309.
Vasudevan, S. V. (1993). Impairment, disability and functional capacity assessment. New York:
The Guildford Press.
Verhagen, A. P., Cardoso, J. R., & Bierma-Zeinstra, S. M. A. (2012). Aquatic exercise and balneo-
therapy in musculoskeletal conditions. Best Practice & Research: Clinical Rheumatology, 26,
335–343.
Viidik, A. (1972). Simultaneous mechanical and light microscopic studies of collagen fibers. Z
Anat Entwicklungsgeasch, 136, 204–212.
Vyazovskiy, V. V., & Harris, K. D. (2013). Sleep and the single neuron: The role of global slow
oscillations in individual cell rest. Nature Reviews. Neuroscience, 14, 443–451.
Wang, T.-J., Belza, B., Thompson, F. E., Whitney, J. D., & Bennett, K. (2001). Effects of aquatic
exercise on flexibility, strength and aerobic fitness in adults with osteoarthritis of the hip or
knee. Journal of Advanced Nursing, 57, 141–152.
Warren, G. L., Lowe, D. A., & Armstrong, R. B. (1999). Measurement tools used in the study of
eccentric contraction-induced injury. Sports Medicine, 27, 43–59.
Wyon, M., Felton, L., & Galloway, S. (2009). A comparison of 2 stretching modalities on
lower-limb range of motion measurements in recreational dancers. Journal of Strength and
Wyon, M., Smith, A., & Koutedakis, Y. (2013). A comparison of strength and stretch interventions
on active and passive ranges of movement in dancers: A randomized controlled trial. Journal of
Strength and Conditioning Research, 27, 3053–3059.
Chapter 9
Conclusion
The detection and response of the cells of the human body to a load induced stimulus
(i.e., stretching), is critical for the response of their internal environment to any
changes from the external environment. As mentioned in previous sections, many
aspects of cellular physiology are integrated with this reaction. The molecular
pathway(s) by which the magnitude of any mechanical perturbation is transmitted,
and associated with changes in skeletal muscle, involves adhesion molecules (dys-
troglycan, integrin, etc.), and the extracellular matrix. In addition, involvement of the
molecules and structures of the contractile apparatus (thick and thin filaments,
M-and Z-discs, titin, obscurin etc.), the sarcolemma, and the cytoskeleton also occur.
The reaction to a mechanical force or load throughout the hierarchies of structures,
from the macroscopic to the microscopic, and how this force is transduced into a
biochemical and functional response, alludes to the concept mechanotransduction.
Stretching intensity within this manuscript references a mechanotransducive
mechanism responsible for evoking a mechanoresponse from the cells of the muscle
and connective tissue to the load or force induced by stretching. The magnitude of
stretching intensity (low, moderate, and high) was investigated to determine if it is
a stimulus for acute inflammation as well as for recovery from muscle damage.
Results from the primary and secondary statistical analysis suggest that high-inten-
sity passive static stretching was likely responsible for acute inflammation, as sug-
gested by increases in hsCRP levels compared to control condition (study one), and
to low- (30% maximum ROM) and moderate (60% maximum ROM) (study two)
stretching intensity, as well as prolonging recovery (study three). In contrast, low-
intensity passive static stretching was not observed to be associated with inflamma-
tion (study two) while at the same time promoting quicker recovery from
unaccustomed eccentric exercise, as measured by perceived muscle soreness and
muscle function (eccentric and isometric peak torque). However, some limitations
pertaining to the methodological design of the three individual studies of the project
do not allow definitive conclusions to be drawn. Given that low-intensity passive
static stretching may have significant beneficial practical applications in both sport
and rehabilitation, further research is required in this area.

Section One: Forms
uestionnaire for Potential Participants in Projects Involving

Q
Blood Analysis
All information given will be treated with strictest confidentiality. Answer all questions by “ticking” the
appropriate box.
1. Are you receiving any medicines and dental treatment, and have you had a recent illness or attending hospital
outpatients? Yes No Not Known
2. Have you had any body piercing or acupuncture, or have you been tattooed in the last 6 months?
Yes No Not Known
3. Have you ever been advised by a doctor not to give blood?
Yes No Not Known
4. Are you or have you ever suffered from any of the following?
Allergies (hay fever, asthma, etc.) ---Yes No Not Known
Anaemia or other blood disorders ---Yes No Not Known
Brucellosis ---Yes No Not Known
Cancer ---Yes No Not Known
Diabetes ---Yes No Not Known
Epilepsy (fits) ---Yes No Not Known
Glandular fever (in last 2 years) ----Yes No Not Known
Heart disease ---Yes No Not Known
Hepatitis (jaundice) or been in contact with a case in the last 6 months --Yes No Not Known
High blood pressure (except during pregnancy) ---Yes No Not Known
Kidney disease ---Yes No Not Known
Peptic ulcers ---Yes No Not Known
Stroke ---Yes No Not Known
Thyroid disease (goitre, etc.) ---Yes No Not Known
Tropical disease especially malaria ---Yes No Not Known
Venereal disease ---Yes No Not Known
5. Is your lifestyle likely to place you at risk of HIV infection (AIDS)? Yes No Not Known
6. Please advise the test supervisor if you have travelled outside Europe within the last 6 months and/or received
travel vaccinations.
Declaration
I have had explained to me, and fully understand, the reasons for blood analysis during exercise testing.
I have not answered Yes to any of the questions listed and to the best of my knowledge am fully eligible to undertake
blood testing and do so of my own free will.
If you have answered Yes to any of the above questions please see test supervisor.
Signature of Participant: ______________________________________________Dated: _______________
I hereby declare that I have read this form in its entirety and I understand that the questions asked have been answered
to my satisfaction.
Signature of Tester: ______________________________________

A Paradigm Shift, https://doi.org/10.1007/978-3-319-96800-1
212 Section One: Forms
Pre-test Questionnaire
Name: _______________________________________________________________________________
Date of Birth: ______________________________________ Age: ________________
As you are to be a participant in this laboratory, would you please answer the following questions truthfully and
completely? The purpose of this questionnaire is to ensure that you are in a fit and healthy state to complete the
exercise test(s). Your cooperation in this is greatly appreciated. (*) Please circle where appropriate
ANY INFORMATION CONTAINED HEREIN WILL BE TREATED AS CONFIDENTIAL.
1. Activity habits
How would you describe your present level of activity*?
Sedentary Moderately active Active Highly active
How would you describe your present level of fitness*?
Unfit Moderately-fit Trained Highly trained
How would you consider your present body weight*?
Underweight Ideal weight Slightly overweight Very overweight
2. Smoking habits
Currently a smoker Yes/No*. A previous smoker Yes/No*, if YES number/day? _____. If previous smoker,
how long since stopping (years)? _____
An occasional smoker Yes/No*, if YES, number/day? _____
A regular smoker Yes/No*, if YES, number/day? _____
3. Consumption of alcohol
Do you drink alcoholic drinks? Yes/No*, if YES, then do you have the occasional drink? Yes/No*
Do you have a drink every day? Yes/No*, have more than one drink a day? Yes/No*
4. Have you had to consult your doctor within the last 6 months? Yes/No*, if YES, please give details to the test
supervisor
5. Are you presently taking any form of medication? Yes/No*, if YES, please give details to the test supervisor
6. Do you suffer, or have you ever suffered from: Asthma? Yes/No*, Diabetes? Yes/No*, Bronchitis? Yes/No*
Epilepsy? Yes/No*, High blood pressure? Yes/No*
7. Do you suffer or have you ever suffered from any form of heart complaint? Yes/No*, if YES, please give
details to the test supervisor
8. Is there history of heart disease in your family? Yes/No*, if YES, please give details to the test supervisor
9. Do you currently have any form of muscle or joint injury? Yes/No*, if YES, please give details to the test
supervisor
10. Have you had any cause to suspend your normal training in the last 2 weeks? Yes/No*, if yes, please give
details to the test supervisor
11. Is there anything to your knowledge that may prevent you from successfully completing the tests that have
been outlined to you? Yes/No*, if YES, please give details to the test supervisor
I have read, understood and completed this questionnaire. Any questions I had were answered to my full satisfaction.
Signature of Participant: ______________________________________________ Date: _____________________
I hereby declare that I have read this form in its entirety and I understand that the questions asked have been
answered to my satisfaction.
Signature of Tester: ________________________________________________________
Section One: Forms 213
tretching Exercises for Study 3: Low-Intensity Passive Static

S
Stretching
Exercise #1: Hamstring
How to perform stretching exercise

Place the left leg on the bench as shown in the diagram with a pillow underneath the left knee.
With the right leg placed perpendicular to the bench, move the upper body forward from the hip
towards the left knee with hands on either side. Hold the GENTLE stretch for FULL 60 s, and
then switch sides. REPEAT each stretch 3 times per side, for a total of 6 min
[Note: To lessen the intensity of the stretch you could move the upper body backward, place a
higher pillow underneath the knee, or move the leg nearest the floor slightly forward]
Parameters of stretching exercise
INTENSITY 30–40% of a
maximum perceived
stretch [warm gentle
feeling]
DURATION 60 s
FREQUENCY 3 times per muscle
group once per day
Diagram
Exercise #2: Hip Flexor

Start with both knees on the ground making sure that your hips and shoulders are square and
that your knees are shoulder width apart. Place a chair in support of the hip flexor that is going
to be stretched. In the diagram, this will be the right side. Place your left hand on the chair.
While supporting yourself with your left hand, extend your right leg away from your body.
Make sure your lower back and upper body are straight and you are not leaning forward. From
this position, lower your body to the ground making sure that your left leg forms a 90° angle at
your knee. You should begin to feel a GENTLE stretch in the right upper thigh hip region. Hold
the stretch for FULL 60 s, and then switch legs. REPEAT each stretch 3 times per side, for a
total of 6 min
INTENSITY 30–40% of a maximum
perceived stretch [warm
gentle feeling]
DURATION 60 sec
FREQUENCY 3 times per muscle group
once per day
Diagram
Exercise #3: Quadriceps

Start on your left side with your body in the position as shown in the diagram below. Make sure
that your right upper leg is resting on a pillow. Grab your right foot with your right hand, and
begin to pull the lower leg towards your right hamstring until you begin to feel a GENTLE
stretch in the right quadriceps. Make sure that your right hip is positioned over the left hip
during the stretch insuring a proper quadriceps stretch. Hold the stretch for FULL 60 s. Switch
sides, and repeat the stretch for the left quadriceps. REPEAT each stretch 3 times per side, for a
total of 6 min
perceived stretch [warm
gentle feeling]
DURATION 60 s
FREQUENCY 3 times per muscle
group once per day
Diagram
Soreness Questionnaire
Could you please CIRCLE THE NUMBER BELOW that best describes your
SORENESS LEVEL each day following the unaccustomed eccentric exercise and
your stretching session? To determine this you need to apply pressure on middle of
the muscle belly of your quadriceps muscle with your fingers.
POST-ECCENTRIC EXERCISE
1 2 3 4 5 6 7 8 9 10
NO SORENESS SORE VERY-VERY SORE
DAY ONE (24 h post-eccentric exercise)

1 2 3 4 5 6 7 8 9 10
DAY TWO (48 h post-eccentric exercise)

1 2 3 4 5 6 7 8 9 10
DAY THREE (72 h post-eccentric exercise)

1 2 3 4 5 6 7 8 9 10
tretching Exercises for Study 3: High-Intensity Passive Static

S
Stretching
Exercise #1: Hamstring

Place the left leg on the bench as shown in the diagram with a pillow underneath the left knee.
With the right leg placed perpendicular to the bench, move the upper body forward from the hip
towards the left knee with hands on either side. Hold the INTENSE stretch for FULL 60 s, and
then switch sides. REPEAT each stretch 3 times per side, for a total of 6 min
[Note: To lessen the intensity of the stretch, you could move the upper body backward, place a
higher pillow underneath the knee, or move the leg nearest the floor slightly forward]
INTENSITY 70–80% of a maximum perceived
stretch [discomfort/slight pain]
DURATION 60 s
FREQUENCY 3 times per muscle group once per
day
Diagram
Exercise #2: Hip Flexor

Start with both knees on the ground making sure that your hips and shoulders are square and
that your knees are shoulder width apart. Place a chair in support of the hip flexor that is going
to be stretched. In the diagram, this will be the right side. Place your left hand on the chair.
While supporting yourself with your left hand, extend your right leg away from your body.
Make sure your lower back and upper body are straight and you are not leaning forward. From
this position, lower your body to the ground making sure that your left leg forms a 90° angle at
your knee. You should begin to feel an INTENSE stretch in the right upper thigh hip region.
Hold the stretch for FULL 60 s, and then switch legs. REPEAT each stretch 3 times per side,
for a total of 6 min
perceived stretch
[discomfort/slight pain]
DURATION 60 s
once per day
Diagram
Exercise #3: Quadriceps

Start on your left side with your body in the position as shown in the diagram below. Make sure
that your right upper leg is resting on a pillow. Grab your right foot with your right hand, and
begin to pull the lower leg towards your right hamstring until you begin to feel an INTENSE
stretch in the right quadriceps. Make sure that your right hip is positioned over the left hip
during the stretch insuring a proper quadriceps stretch. Hold the stretch for FULL 60 s. Switch
sides, and repeat the stretch for the left quadriceps. REPEAT each stretch 3 times per side, for a
total of 6 min
perceived stretch
[discomfort/slight pain]
DURATION 60 s
once per day
Diagram
Soreness Questionnaire
Could you please CIRCLE THE NUMBER BELOW that best describes your
SORENESS LEVEL each day following the unaccustomed eccentric exercise and
your stretching session? To determine this you need to apply pressure on middle of
the muscle belly of your quadriceps muscle with your fingers.
POST-ECCENTRIC EXERCISE
1 2 3 4 5 6 7 8 9 10
DAY ONE (24 h post-eccentric exercise)

1 2 3 4 5 6 7 8 9 10
DAY TWO (48 h post-eccentric exercise)

1 2 3 4 5 6 7 8 9 10
DAY THREE (72 h post-eccentric exercise)

1 2 3 4 5 6 7 8 9 10
Section Two: Samples of Raw Data for Studies
Study 1
Table A.1 Age, mass, height, and hsCRP values

hsCRP − high-intensity
passive static stretching
hsCRP − control (mg/L) (mg/L)
Age Mass hgt Immediately 24 h Immediately 24 h
Participants (years) (kg) (m) Pre post post Pre post post
1 33 92 1.78 0.92 0.95 0.92 1.53 2.67 4.52
2 24 83 1.83 0.71 – 0.71 2.17 2.20 –
3 35 76 1.71 1.13 1.19 1.13 0.48 0.69 7.08
4 29 86 1.86 0.78 0.83 0.78 0.78 1.25 1.83
5 25 95 1.85 – 4.64 – 1.03 1.14 1.19
6 23 79 1.70 0.74 0.81 0.74 0.57 0.74 0.81
7 31 68 1.70 1.80 – 1.80 4.51 7.22 10.00
8 33 71 1.72 0.86 0.69 0.86 1.38 1.41 1.57
9 34 83 1.73 0.63 1.30 0.63 0.99 0.99 1.34
10 25 78 1.77 1.22 0.70 1.22 1.17 1.42 1.79
11 28 69 1.73 1.03 0.64 1.03 0.82 0.94 5.73
12 25 72 1.73 0.33 0.76 0.33 0.36 1.40 3.20

222 Section Two: Samples of Raw Data for Studies
Study 2
Table A.2 Age, mass, height, and hsCRP values for 30%, 60%, and 90% maximum ROM
30% maximum 60% maximum 90% maximum
ROM ROM ROM
Age Mass hgt 24 h 24 h 24 h
Participant (years) (kg) (m) Pre Post post Pre Post post Pre Post post
1 22 99 1.91 0.97 0.91 0.53 1.01 0.98 0.95 1.55 1.67 3.83
2 23 77 1.70 0.68 0.71 0.58 0.47 0.44 0.64 1.41 1.29 0.71
3 27 91 1.79 0.26 0.27 0.42 1.23 1.24 0.83 1.51 1.44 1.20
4 20 89 1.83 1.09 0.97 1.08 1.07 1.04 0.80 2.99 3.34 3.89
5 32 94 1.80 0.80 0.75 0.45 1.10 1.14 1.20 0.49 0.56 0.96
6 25 88 1.83 0.28 0.27 0.57 0.20 0.22 0.29 4.54 4.90 2.43
7 41 72 1.75 0.31 0.28 0.39 0.69 0.68 0.61 0.31 0.33 0.38
8 21 87 1.78 0.59 0.64 0.45 0.67 0.71 1.36 3.14 3.57 2.62
9 25 87 1.76 1.61 1.70 1.84 1.62 1.61 1.32 5.81 5.87 9.90
10 20 82 1.76 0.25 0.28 0.24 0.52 0.47 0.46 0.28 0.28 0.42
11 25 73 1.73 0.36 0.41 0.38 0.37 0.37 0.50 0.51 0.47 0.54
h hours, ROM range of motion
Study 3
Table A.3 Perceived muscle soreness and eccentric and isometric peak torque
Perceived Eccentric peak torque Isometric peak
muscle soreness (Nm) torque (Nm)
Time (h) Time (h) Time (h)
Conditions n 0 24 48 72 0 24 48 72 0 24 48 72
Low-intensity passive 1 7 6 2 1 316 310 326 332 272 268 275 275
static stretching (LIS) 2 8 6 3 1 279 217 222 273 154 162 177 186
3 7 6 3 2 182 176 179 176 187 177 207 191
4 5 4 3 2 149 169 176 176 159 128 133 163
5 5 4 4 1 192 170 221 243 187 162 194 207
6 5 5 1 1 314 323 316 360 264 260 296 320
7 8 5 2 1 211 178 210 221 198 180 203 211
8 4 2 2 1 320 307 282 317 238 242 212 255
9 6 6 4 1 237 197 274 270 201 182 218 225
10 5 6 5 1 275 249 236 263 216 203 180 190
(continued)
Section Two: Samples of Raw Data for Studies 223
Table A.3 (continued)
Perceived Eccentric peak torque Isometric peak
muscle soreness (Nm) torque (Nm)
Time (h) Time (h) Time (h)
Conditions n 0 24 48 72 0 24 48 72 0 24 48 72
High-intensity passive 1 1 1 1 1 208 168 223 183 178 147 146 172
static stretching (HIS) 2 7 8 4 2 194 147 168 186 144 114 130 155
3 6 6 3 3 303 188 256 188 222 194 212 221
4 6 6 4 2 321 143 250 200 230 202 216 231
5 6 6 3 2 186 160 178 178 191 161 164 166
6 6.5 7 3 2 173 173 216 204 151 154 161 180
7 6 7 3 1 270 257 238 269 229 204 221 243
8 7 5 5 2 189 204 256 226 208 228 254 220
9 7 8 6 4 136 150 130 163 113 117 112 131
10 7 3 5 5 202 144 165 162 147 114 111 147
Control 1 3 3 3 3 336 288 258 328 314 241 242 252
2 8 8 7 2 207 196 197 214 153 167 198 201
3 6 3 3 1 255 232 176 260 219 214 223 247
4 4 4 3 1 193 175 167 138 172 158 158 138
5 7 8 3 2 216 234 182 216 228 222 209 202
6 7 7 5 3 172 140 174 160 160 144 137 143
7 4 6 3 1 173 140 143 126 128 105 100 94
8 8 7 5 5 195 216 183 212 149 149 155 169
9 7 6 4 1 244 207 218 231 180 165 183 185
10 5 5 4 3 157 134 96 121 148 86 91 97
Nm Newton metres
If you truly want to understand something….you must try to change it

Kurt Lewin
Glossary
ANOVA Analysis of variance

Ca2+ Calcium
CI Confidence interval
“d” Cohen’s d
dys days
DOMS Delayed onset muscle soreness
ECM Extracellular matrix
ED1+ Monocyte/pro-inflammatory macrophage (phagocytic)
ED2+ Anti-inflammatory macrophage (non-phagocytic)
ε (epsilon) Estimating sphericity
hgt Height
hsCRP High sensitivity C-reactive protein
IL- Interleukin (-1β, -1ra (receptor antagonist), -6, -8, -10, -12, -15)
kg Kilograms
km Kilometres
LN Logarithm (natural)
m Metres
mg/L Milligrams per litre
ml Millilitres
Mm Millimetre
μl Micro litre
MPO Myeloperoxidase
mRNA Messenger ribonucleic acid
MTU Myotendon unit
PARQ Physical activity readiness questionnaire
PBS Phosphate buffered solution
PEVK Proline-glutamate-valine-lysine
PNF Proprioceptive neuromuscular facilitation
R Correlation coefficient

226 Glossary
rads/sec Radians per second

RMANOVA Repeated measures analysis of variance
ROM Range of motion
sec Seconds
TGF-β1 Transforming growth factor—β1
TNF-α Tumour necrosis factor—α
U/L Units per litre
Index
A neutrophils, 75
The Achilles Heel, 30–35 skeletal muscle contraction, 75
Acute inflammatory response Z-disc, 72, 73
active tension, 131
acute muscle damage, 132
body position, 131 B
connective and muscular tissues, 131 Ballistic stretching, 10
C-reactive protein, 132, 139, 140
effect sizes
between conditions, 136–138 C
within conditions, 138 Calpain, see Autolytic calpain
IL-6, 140 Cells, 42
intensity, 131 Circulating macrophages, 80
methods Costameres
biomarkers, 135 associations, 32
control intervention, 135 cell-matrix interface, 35
high-intensity passive static stretching dynamic connection, 35
(IS) protocol, 134–135 dystrophin, 32, 33
participants, 132–133 ECM, 30–32
power calculations, 133 hierarchy of interaction, 31
procedures, 133, 134 integral and peripheral membrane, 30
statistical analysis, 135–136 integrins, 34
pro-inflammatory cytokines, 139 ligands, 34
RMANOVA, 136, 137 mechanical linkage, 35
stretch intensity, 132 mechanoresponsiveness, 33
time-dependent activity, 131 musculoskeletal tissues, 31
Acute phase response, 57–59 myofibril, 31
Anti-inflammatory cytokines, 86 physical forces, 34
Autolytic calpain, 69, 75 sarcolemmal structures, 30
basement membrane, 72 structural and regulatory proteins, 31
catabolic events, 72 tensegrity, 30
contraction and relaxation, 73 transmembrane β-dystroglycan, 33
eccentric exercise, 73 vinculin, 33
lesions, 72 vinculin-talin-integrin, 33
MPO, 73, 75 Z-disc, 30, 32

228 Index
Costameres (cont.) thin filament, 19

α-actinin rich Z-discs, 31 third filament, 21–23
β1 sub-family, 35 titin, 19
Creatine kinase (CPK), 155 Z-disc, 25–28
Cytokines, 56, 57, 183, 185, 198, 200 FlowCytomix Simplex Kit, 135
Cytoskeleton, 7 Force, 42–44
Frequency, 51–53
D
Duration, 51–53 G
Dynamic stretching, 11 Glycosaminoglycans (GAGs), 47
Golgi tendon organs (GTO), 6, 131
E
Eccentric peak torque, 186 H
Effectors, 49 Hamstring, 213, 217
Endogenous glucose-producing organ, 63 High-sensitivity C-reactive protein (hsCRP), 58
Extracellular matrix (ECM), 3 Hip flexor, 214, 218
amalgamation, 45 Homeostasis, 5
biological relevance, 46
chaos theory, 44
classification, 45 I
communication, 46 Inflammation
dynamic structure, 45 acute phase response, 57–59
effectors, 49 cardinal signs, 53
elastic properties, 47 cellular and humoral responses, 53
energetic system, 44 characteristic sign, 53
fibroblasts, 47 cytokines, 56, 57
functional requirements, 47 defense mechanism, 53
hierarchical organisation, 45 eccentric exercise, 54
homeostasis, 46 injurious stimulation, 54
macromolecules, 46 injury-specific interaction, 55
magnitude, 46 macrophages, 55
mechanical homeostasis, 48 microcirculation, 54
molecular scaffold, 46 muscle (see Muscle)
regenerative processes, 45 myokines, 62, 63
sensors, 50 neutrophils, 55
stiffness (rigidity)/elasticity, 47 non-damaging exercise, 60, 61
structure and function, 47 responses, 54
substrate, 48, 49 sensors, 54
transmembrane structures, 47 target tissues, 54
Inflammatory cell response, 66, 69, 70
Inflammatory cells, 68, 69, 78, 79, 82, 92–94
F Inflammatory response
Filament systems of skeletal muscle acute inflammatory response, 3
A-band, 18 biological system, 1
concealed protein, 24, 25 chronic inflammation, 2
fourth filament, 23, 24 communication network, 1
I band, 18 complex system, 1
mechanical relay system, 29 ECM, 3
M-line, 19 injury, 3
myofibrillar proteins, 18 mediators, 3
nebulin, 19 mental and emotional stress, 1
thick filament, 20, 21 musculoskeletal tissue, 2
Index 229
pain and/or discomfort, 2 intensity, 64, 65

pain/discomfort, 2 isometric, 15
pathophysiology, 2 (see Stretching mode of exercise, 64, 65
intensity) MTU, 17
stretching parameters, 2 myofibril, 16
synergistic process, 1 post-mitotic tissue, 15
tissue homeostasis and integrity, 2 sarcomere, 16
Integrins, 34 skeletal, 14
Intensity, 51–53, 64, 65 stretching, 15
Interleukin 1 receptor antagonist (IL-1ra), 59 striated, 18
Intermediate filaments, 16, 18, 24–26, 29, 31, Muscle soreness, 222–223
33, 37 Musculoskeletal disorders, 201
International Association for the Study of Musculoskeletal tissue(s), 6
Pain, 2 Myeloperoxidase (MPO), 73, 75
Isometric peak torque, 222–223 Myokines, 62, 63
Myotendon unit (MTU), 6, 7, 9, 17, 36, 38,
40–42, 45, 51, 91
L
Least significant difference (LSD), 136
N
Nebulin, 23, 24
M Neutrophil-endothelial interaction, 62
Macrophages, 54, 55, 70, 76, 78–80 Neutrophils, 55, 78, 79
Mechanical relay system, 29 Non-damaging exercise, 60, 61
Mechanotransducers, 34
Mechanotransduction, 5, 28, 30, 31, 33, 35,
41, 49, 51, 87–91 O
Muscle Obscurin, 24, 25
architecture, 36 Osteoarthritis (OA), 3
ATP, 15
composed of, 16
concentric, 15 P
cytoskeletal elements, 17 Passive static stretching, 132, 134–135,
damage 138–140
autolytic calpain, 69 biochemical injury, 160
comparison, 62 DOMS, 159, 160
cytokine, 66 dose-response effect, 177
four stages, 66 eccentric peak torque, 168–174
pro- or anti-inflammatory, 66 high-intensity passive static stretching vs.
sequence/time response, 65 control, 168, 170–172
stage 1, 70–72 inflammatory cells, 160
stage 2, 72–75 inflammatory response, 159
stage 3, 75–78 isometric peak torque, 177
stage 4, 78–83 low- vs. high-intensity passive static
degeneration and regeneration, 15 stretching, 166
duration, 64, 65 low-intensity passive static stretching vs.
eccentric, 15 control, 168
endogenous glucose-producing organ, 63 magnitude-based inference approach
environmental changes, 15 high-intensity passive static stretching
exo- and endosarcomeric network vs. control, 173, 176
systems, 18 low- vs. high-intensity passive static
filament systems (see Filament systems of stretching, 172, 176
skeletal muscle) low-intensity passive static stretching
G-protein family, 16 vs. control, 173, 176
230 Index
Passive static stretching (cont.) Static stretching, 9, 10

methods Stretching
eccentric exercise protocol, 163 biological systems, 13
maximum perceived stretch, 162 classification, 9–12
methodology schematic of description, 6–8
intervention(s), 162 and inflammatory cells, 92–94
online randomisation programme, 162 internal and external elements, 14
participants, 161 interrelationship(s), 14
passive static stretching exercise in-vitro studies, 13
protocol, 164, 165 in-vivo studies, 13
pre unaccustomed eccentric exercise low and high frequency filters, 14
assessment, 163 magnitude, 14
statistical analysis, 165–166 measurement, 6–8
muscle function, 161, 163, 165, 176–178 physical strategy, 6
muscle soreness, 176 strain factors, 51–53
muscular actions, 160 Stretching intensity, 131, 132
neutrophils, 160 acute and chronic inflammation, 198
non-steroidal anti-inflammatory drugs, 160 acute inflammation, 209
perceived muscle soreness, 166–168 acute inflammatory response, 145,
post-training effects, 159 183–185
pre- and post-exercise static stretching, 161 age, 193
unaccustomed eccentric exercise, 159, 161, blood collection, 189–191, 198
163, 164, 166 calcium channel blocker, 197
Phosphate buffered solution (PBS), 81, 82 calcium-dependent protease, 197
Physical activity readiness questionnaire compliance, 192
(PARQ), 132–133, 146, 161 C-reactive protein, 145, 154, 189
Physical training programmes, 6 cytokines, 189
Pro- and anti-inflammatory macrophages, 79 effect sizes
Proprioceptive neuromuscular facilitation linear regression analysis, 151–153
(PNF), 11, 12 post intervention hsCRP, 150, 151
Proteoglycans (PGs), 47 pre-intervention hsCRP, 151
regression analysis, hsCRP, 152–154
hepatocytes, macrophages and
Q lymphocytes, 189
Quadriceps, 215, 219 high-intensity passive static stretching, 199
hsCRP, 189
IL-6, 145, 189
R IL-6 and creatine kinase, 199
Reactive oxygen species, 200–201 vs. inflammation, 185–186
Rehabilitation protocols, 6 inflammatory response, 183
Repeated measures analysis of variance intensity, 145, 155
(RMANOVA), 135 low- and moderate-intensity passive static
Resident macrophages, 80, 81 stretching, 199
Rheumatoid arthritis (RA), 3 low- vs. high-intensity passive static
stretching, 197
low-, moderate- or high-intensity passive
S static stretch influences, 183
Sarcomere, 7, 16–24, 26, 28, 31, 32, 36, 40, low-intensity passive static stretching, 209
41, 54, 70–72, 90, 94 maximum ROM, 145, 154
Sarcomeric proteins, 20, 22, 35 mechanical force, 145, 155
Self-regulation, 5 mental stress, 193
Sensors, 50 method
Soreness levels, 216, 220 blood biomarkers, 149
Spatial sequencing, 81–85 C-reactive protein, 149
Stability, balance, and control (SBC), 132 experimental Set-Up, 147
Index 231
Kin Com, 148 cytoskeletal proteins, 41

knee immobiliser, 146, 147 dystrophin-glycoprotein complex, 40, 41
maximal physical pain/effort, 147 ECM, 37
methodology schematic of finger-like projections, 40
intervention(s), 148 force transmission, 40
participants, 146 integrated mechanical unit, 41
statistical analysis, 149–150 interaction, 37
methodology, 191 interfacial folding, 40
moderate and high-intensity stretching, 197 junctional membrane, 41
muscle tissue, 154 load-bearing tissue, 37
musculoskeletal disorders and pain, 201 loading period, 38
musculoskeletal system, 198 magnitude, 38
nutrition, 192 MTU, 39, 41
passive static stretching intensity, 186 sarcolemma, 40
pro-inflammatory cytokines, 198 sarcomeric forces, 38
quantitative, 155 structural hierarchy, 37
recovery and regeneration, 202–203 teno-osseous junction, 37
relaxation response, 203–204 tissue structure, 38
RMANOVA, 150–151 transmission, 39
ROS, 200–201 Tensegrity, 30, 88, 91
secondary analysis, 155 Tissue, 42
sensation magnitudes, 155 Titin, 21–23
stretching intensity, 155 Transendothelial migration (TEM), 69, 75–78
ultrasonography, 199–200 Transformational processes, 5
Stretching magnitude, 5, 7, 10, 14, 19, 34, 42,
52, 83, 90, 91
Substrate, 48, 49 U
Sympathetic nervous system (SNS), 203 Ultrasonography, 199–200
Unaccustomed eccentric exercise, 160
University of Wolverhampton’s ethics
T committee, 146
Tendons, 6, 7, 10, 30, 49–51, 81–83, 89, 90, 93
basal lamina, 40
communication, 40 Z
cylindrical skeletal muscle fibres, 38 Z-disc, 18, 25–28

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Nikos C. Apostolopoulos - Stretch Intensity and The Inflammatory Response - A Paradigm Shift-Springer International Publishing (2018)

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Nikos C.

ISBN 978-3-319-96799-8 ISBN 978-3-319-96800-1 (eBook)

Library of Congress Control Number: 2018961999

© Springer Nature Switzerland AG 2018

“The true nature of knowledge is experiment…

Toronto, ON, Canada Nikos C. Apostolopoulos

3 Study One: Acute Inflammatory Response to Stretching�������������������� 131

Perceived Muscle Soreness�������������������������������������������������������������������� 166

Section One: Forms������������������������������������������������������������������������������������������ 211

Section Two: Samples of Raw Data for Studies�������������������������������������������� 221

Fig. 2.1 Classification of stretching������������������������������������������������������������������ 9

Fig. 2.25 Stage 4: Inflammatory cells—pro-inflammatory macrophages

Table 2.1 Time scale of inflammatory cell response in relation

Table A.1 Age, mass, height, and hsCRP values���������������������������������������������� 221

© Springer Nature Switzerland AG 2018 1

Inflammation can be either acute or chronic, but in reality it represents a

An injured muscle typically undergoes stages of degeneration, inflammation,

© Springer Nature Switzerland AG 2018 5

The Definition of Stretching and Its Measurement

Stretching, a movement applied by an external and/or internal force stressing con-

Stretching is classified as static, dynamic, and pre-contraction (Fig. 2.1). Within

a controversy exists whether this change is due to a decrease in MTU stiffness

combination with other techniques presents a more appropriate approach with

Proprioceptive Neuromuscular Facilitation (PNF)

This stretching technique developed to improve muscle elasticity to treat neurologi-

 acroscopic and Microscopic Information Processing Levels:

In vitro studies have been instrumental in discovering, observing, and determining

Muscles, Tendons, and MTU

importantly, the relaying of information from one sarcomere to another. These

The Filament Systems of Skeletal Muscle

Thin Filament (Actin, Troponin, Tropomyosin, and Tropomodulin) (Fig. 2.3)

Thick Filaments (Myosin)

Third and Fourth Filaments and Obscurin

Titin (Connectin) (Third Filament): The Titan Amongst Proteins

domain is the only region exhibiting elastic stretch dependence, observed to

Nebulin (Fourth Filament): The Uncertain Protein

Obscurin: The Concealed Protein

studies, removal of obscurin was associated with changes in the longitudinal

Z-Disc, Intermediate Filaments, and Costameres

The primary function of the proteins/structures of the cytoskeleton of the muscle

as a passive structure of the sarcomere linking actin filaments of opposite polarity

its flexibility, accommodating changes in cell geometry in response to muscle

Intermediate Filaments: The Mechanical Relay System

Costameres: The Achilles Heel

Fig. 2.9 Costamere associations. (Published with kind permission of copyright © Nikos

As suggested by sections above, a complex dynamic interaction exists between the

Myotendon Unit (MTU) (Fig. 2.10)

Fig. 2.10 Myotendon unit. (Published with kind permission of copyright © Nikos

of this interfacial folding by investigators have interpreted this folding as an

 acroscopic and Microscopic Information Processing Levels:

Force, Cells, and Tissue

Forces have been increasingly recognised as major regulators of cell structure

The Extracellular Matrix (ECM)

component parts interacting in a non-linear manner. The information processed

The stiffness (rigidity) or elasticity of the ECM is an important material property

mechanical homeostasis of the matrix, producing and secreting the matrix

 he Strain Factors of Stretching: Intensity, Duration,

Stretching initiates a stress/strain response on the connective tissue dependent on its

Toronto, ON, Canada Nikos C. Apostolopoulos

3 Study One: Acute Inflammatory Response to Stretching�� 131

Perceived Muscle Soreness�� 166

Section One: Forms�� 211

Section Two: Samples of Raw Data for Studies�� 221

Fig. 2.1 Classification of stretching�� 9

Table A.1 Age, mass, height, and hsCRP values�� 221

The Definition of Stretching and Its Measurement

Proprioceptive Neuromuscular Facilitation (PNF)

acroscopic and Microscopic Information Processing Levels:

Muscles, Tendons, and MTU

Myotendon Unit (MTU) (Fig. 2.10)

acroscopic and Microscopic Information Processing Levels:

Force, Cells, and Tissue

The Extracellular Matrix (ECM)

he Strain Factors of Stretching: Intensity, Duration,

eutrophils, Macrophages, Cytokines, and Acute Phase

Acute Phase Response

Non-damaging Exercise and the Inflammatory Response

Damaging Exercise and the Inflammatory Response

Stretching and Inflammatory Cells

Participants were randomised into either an IS or control group, using a computer-

High-Intensity Passive Static Stretching (IS) Protocol

Effect Sizes Between Conditions

Effect Sizes Within Conditions

Post Intervention hsCRP

Post Intervention hsCRP: 24 h

econdary Analysis: Linear Regression (30–90 and 60–90%