Eur J Appl Physiol
DOI 10.1007/s00421-013-2693-9
ORIGINAL ARTICLE
Effect of cryotherapy on muscle recovery and inflammation
following a bout of damaging exercise
Naomi J. Crystal • David H. Townson •
Summer B. Cook • Dain P. LaRoche
Received: 18 July 2012 / Accepted: 3 July 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract The purpose of this study was to determine the
effect of cryotherapy on the inflammatory response to
muscle-damaging exercise using a randomized trial.
Twenty recreationally active males completed a 40-min run
at a -10 % grade to induce muscle damage. Ten of the
subjects were immersed in a 5 °C ice bath for 20 min and
the other ten served as controls. Knee extensor peak torque,
soreness rating, and thigh circumference were obtained
pre- and post-run, and 1, 6, 24, 48, and 72 h post-run.
Blood samples were obtained pre- and post-run, and 1, 6
and 24 h post-run for assay of plasma chemokine ligand 2
(CCL2). Peak torque decreased from 270 ± 57 Nm at
baseline to 253 ± 65 Nm post-run and increased to
295 ± 68 Nm by 72 h post-run with no differences
between groups (p = 0.491). Soreness rating increased
from 3.6 ± 6.0 mm out of 100 mm at baseline to
47.4 ± 28.2 mm post-run and remained elevated at all
time points with no differences between groups
Communicated by William J. Kraemer.
N. J. Crystal
S. B. Cook
D. P. LaRoche
Robert Kertzer Exercise Physiology Laboratory,
University of New Hampshire, Durham, NH, USA
e-mail: NaomiJCrystal@gmail.com
S. B. Cook
e-mail: Summer.Cook@unh.edu
N. J. Crystal
S. B. Cook
D. P. LaRoche (&)
Department of Kinesiology, University of New Hampshire,
124 Main St, Durham, NH 03824, USA
e-mail: Dain.LaRoche@unh.edu
D. H. Townson
Department of Molecular, Cellular, and Biomedical Sciences,
University of New Hampshire, Durham, NH 03824, USA
e-mail: Dave.Townson@unh.edu
(p = 0.696). CCL2 concentrations increased from
116 ± 31 pg mL-1 at baseline to 293 ± 109 pg mL-1 at
6 h post-run (control) and from 100 ± 27 pg mL-1 at
baseline to 208 ± 71 pg mL-1 at 6 h post-run (cryother-
apy). The difference between groups was not significant
(p = 0.116), but there was a trend for lower CCL2 in the
cryotherapy group at 6 h (p = 0.102), though this measure
was highly variable. In conclusion, 20 min of cryotherapy
was ineffective in attenuating the strength decrement and
soreness seen after muscle-damaging exercise, but may
have mitigated the rise in plasma CCL2 concentration.
These results do not support the use of cryotherapy during
recovery.
Keywords Eccentric exercise
Downhill run

Inflammation
Chemokine ligand-2
Abbreviations
CCL2 Plasma chemokine ligand 2
DOMS Delayed-onset muscle soreness
MVC Maximal voluntary contraction
VASS Visual analog scale for soreness
Introduction
Cryotherapy, commonly performed by way of ice baths, is
a popular post-exercise recovery modality utilized by ath-
letes and clinicians to reduce inflammation and speed
recovery (Barnett 2006; Cheung et al. 2003; Connolly et al.
2003). In the context of this paper, cryotherapy is defined
as immersion in water below 15 °C (Bleakley and Davison
2010). While there have been investigations of the effects
of cryotherapy on inflammation and recovery from
123

damaging exercise (Eston and Peters 1999; Howatson et al.
2009; Ingram et al. 2009; Jakeman et al. 2009; Lane and
Wenger 2004; Paddon-Jones and Quigley 1997; Rowsell
et al. 2009; Sellwood et al. 2007; Vaile et al. 2008), there is
no clear consensus on the effectiveness of this treatment
modality, despite its widespread use.
Exercise induces an inflammatory response proportional
to the duration, intensity, and mass of muscle used as well
as the damage resulting from the exercise (Camus et al.
1993; Giraldo et al. 2009). To study muscle impairment
and inflammation in a laboratory setting, eccentric exercise
is commonly used as a means of inducing muscle injury.
Running, particularly downhill running, has a large
eccentric component and produces significant myocyte
damage and inflammatory response (Buford et al. 2009;
Malm et al. 2004; Smith et al. 2007).
Myocyte damage is manifested by delayed-onset muscle
soreness (DOMS), strength and power decrements, as well
as an increase in plasma concentrations of proteins nor-
mally found within the muscle cell (e.g., creatine kinase)
(Best and Hunter 2000). An acute inflammatory response
ensues following this damage including the release of
inflammatory cytokines (Best and Hunter 2000). The
inflammatory response is accompanied by symptoms such
as swelling and soreness, which individuals may seek to
attenuate with cryotherapy (Best and Hunter 2000). The
inflammatory response to downhill running (Hubal et al.
2008; Peake et al. 2005) and other eccentric exercises is
well characterized (Malm et al. 2000), and has recently
been reviewed by Tidball (2005).
An inflammatory cytokine of interest is chemokine
ligand 2 (CCL2) because it is a sensitive marker of
inflammation that increases after running (Peake et al.
2005). Furthermore, it shows reduced levels after a second
session of exercise, indicating that it may be involved in
the repeated bout effect, or adaptation to eccentric exercise
(Smith et al. 2007). CCL2 serves to recruit monocytes to
the damaged tissue so they can phagocytize cellular debris
and facilitate the rebuilding process (Chazaud et al. 2009).
Research in CCL2-deficient mice has demonstrated that the
cytokine is necessary for the recruitment of monocytes for
phagocytosis and stimulates production of insulin-like
growth factor 1 (IGF-1) for repair (Lu et al. 2011; Shir-
eman et al. 2006). Although it would negatively impact
adaptation, a reduction in CCL2 may be beneficial for
short-term recovery in that it would attenuate secondary
damage caused by the inflammatory process and the
associated DOMS, thus potentially mitigating the perfor-
mance decrement.
Though immune cells repair exercise-induced muscle
damage, they also exacerbate the injury by releasing
reactive oxygen species (ROS) that oxidize molecules
123
Eur J Appl Physiol
within the myocyte (Best et al. 1999; Brickson et al. 2001).
This results in an increase in the noticeable symptoms of
muscle damage including DOMS and strength decrement.
Because these symptoms interfere with subsequent com-
petition and training performance, elite and recreational
athletes often seek strategies to minimize muscle damage
and speed recovery (Cheung et al. 2003). Recovery strat-
egies such as stretching, massage, light activity, and
cryotherapy are purported to reduce inflammation, dimin-
ish soreness, and facilitate a more rapid return of perfor-
mance capabilities (Ingram et al. 2009; Lane and Wenger
2004; Lapointe et al. 2002; Tidball and Wehling-Henricks
2007; Vaile et al. 2008). Speeding the recovery process is
especially beneficial when applied to occasions when an
athlete must perform with little recovery time between
competitions. It is also valuable for the general exerciser,
as unaccustomed exercise causes soreness and loss of
function which can interfere with daily activities and sub-
sequent exercise bouts.
Studies investigating cryotherapy as a recovery modality
have used a range of immersion protocols and have mea-
sured different markers of muscle damage making it dif-
ficult to assess efficacy. Some studies have found
cryotherapy to be effective in reducing the symptoms of
muscle damage such as swelling, soreness, sprinting per-
formance impairment, cycling time trial and interval per-
formance impairment, and elevated plasma creatine kinase
and C-reactive protein (Eston and Peters 1999; Ingram
et al. 2009; Lane and Wenger 2004; Rowsell et al. 2009;
Vaile et al. 2008). However, other studies indicate that
cryotherapy has no effect on performance, soreness,
swelling, plasma creatine kinase, or lactate dehydrogenase
measures (Howatson et al. 2009; Jakeman et al. 2009;
Paddon-Jones and Quigley 1997; Rowsell et al. 2009;
Sellwood et al. 2007). Small sample sizes, small effect
sizes, and high variability of measures among subjects may
have prevented statistical significance in these studies, yet
it is plausible that cryotherapy is ineffective at aiding
muscle recovery. Subsequently, there is no agreement on
the effectiveness of cryotherapy as an exercise recovery
modality.
The purpose of this study was to examine the impact of
cryotherapy on the inflammatory response to downhill
running, and muscle recovery over 3 days, by analyzing
plasma levels of CCL2 as well as the more commonly
studied soreness, swelling, and isometric strength vari-
ables. It was hypothesized that cryotherapy would reduce
the inflammatory response to downhill running, specifically
that cryotherapy would attenuate the rise in CCL2 normally
observed following downhill running, ameliorate DOMS,
reduce swelling in the thigh, and minimize knee extensor
strength decrement.
Eur J Appl Physiol
Methods
Subjects
Twenty males (mean ± SD: age 21.2 ± 2.3 years; height
1.78 ± 0.05 m; mass 76.4 ± 9.6 kg; VO2peak = 58.9 ±
8.6 ml kg-1 min-1), who were unaccustomed to cryother-
apy, were recruited to the study via flyers around the university
and by word of mouth. Subjects were then randomly assigned
to the cryotherapy or control group (n = 10 per group).
Subjects were classified as recreationally active from a self-
reported physical activity questionnaire. We defined ‘‘recre-
ationally active’’ as meeting the American College of Sports
Medicine’s guidelines for cardiovascular exercise, that is,
30 min of moderate intensity (3–5.9 METs) exercise 5 days
per week or 20 min of vigorous (C6 METs) exercise 3 days
per week, but not exceeding 7 h of vigorous exercise per
week. Subjects reported a diverse history of training which
included recreational exercise, as well as previous participa-
tion in high school and college athletics, but at the time of the
study all met our definition of recreationally active. Subjects
also reported a variety of activities in which they routinely
engaged including running, team sports, strength training,
cycling, and cross-country skiing. Subjects were excluded
from the study for having any musculoskeletal injuries that
interfered with running, having Reynaud’s disease or cold
allergy, or regularly using anti-inflammatory medication. One
subject withdrew from the study 5 min into the downhill run
and an additional subject was added in his place. The Uni-
versity of New Hampshire’s Institutional Review Board
approved the use of human subjects in accordance with the
Belmont Report, and written informed consent of each subject
was obtained prior to their participation.
Experimental design
The study was a randomized clinical trial that evaluated the
effect of cryotherapy (independent variable) on the
Fig. 1 Timeline of the study. Pre-run immediately before the downhill run, Post-run immediately after the cooldown following the downhill run
inflammatory cytokine CCL2, isometric knee extensor
strength, perceived soreness, and swelling (dependent
variables). These dependent variables were chosen as they
are markers of muscle damage, they are commonly reported
symptoms of eccentric exercise, they may impact physical
performance, and they are reasons for which individuals
would use cryotherapy. The study took place from October
to June. Subjects visited the laboratory six times and a
timeline summary of the study can be seen in Fig. 1. Visit 1
consisted of anthropometric measurements, a peak oxygen
consumption (VO2peak) test, determination of downhill
running speed, and familiarization sessions with the
strength assessment and visual analog scale for soreness
(VASS). Visit 2 took place an average of 9 ± 6 days after
the first visit and each session began between 11:00 a.m.
and 1:00 p.m. to account for diurnal variation. A blood
draw for assay of CCL2, and baseline strength, thigh cir-
cumference, and soreness data were collected prior to the
downhill run. Subjects then completed a 40-min downhill
run to induce muscle damage and all measures were
recorded again post-run. The cryotherapy (ice bath) or
control condition was completed immediately after post-run
measures were taken and all measures were recorded again
at 1, 6 h (Visit 3), and 24 h (Visit 4) after completion of the
downhill run. At 48 h (Visit 5) and 72 h (Visit 6) after the
run, only strength, thigh circumference, and soreness mea-
sures were collected as CCL2 was expected to return to
baseline by 24 h post-exercise (Smith et al. 2007).
VO2peak testing and determination of individual
downhill running speed
During Visit 1 a modified Balke protocol was used to
determine VO2peak during treadmill running (Quinton, Q65,
Seattle, WA, USA). Subjects began running at a moderate
running pace (between 2.7 and 3.8 m s-1 based on self-
reported fitness level) at 0 % grade; then the grade was
increased 1 % per minute until volitional exhaustion.
123

Continuous respiratory measurements were recorded using
a SensorMedics Vmax Metabolic Measurements Cart
(CareFusion Corporation, San Diego, CA, USA). Data
were recorded breath by breath and averaged over 30 s for
analysis. Heart rate was monitored with a PolarTM heart
rate monitor (Polar Electro, Lake Success, NY, USA) and
recorded at the end of each minute. Ratings of perceived
exertion (RPE) on the 6–20 point Borg scale were recorded
at the end of each stage.
To determine the appropriate intensity for the downhill
runs, 60 % of VO2peak was calculated for each participant.
The familiarization session with downhill running also
served as a test to determine the treadmill speed necessary
to achieve an intensity of 60 % VO2peak while running at a
-10 % grade on the treadmill, and was completed during
Visit 1. Subjects began jogging at 1.7–2.5 m s-1 on a level
treadmill and the grade was gradually lowered to reach a
-10 % grade. Oxygen consumption data were collected
and speed was increased until oxygen consumption reached
60 % of VO2peak. This speed was recorded and used during
the downhill run on Visit 2. Subjects ran downhill for no
longer than 5 min during the habituation session to avoid
inducing DOMS and a repeated bout effect.
Exercise protocol
Subjects were instructed to refrain from any vigorous
exercise for at least 72 h before the downhill run and until
after the 72 h post-run visit. Subjects were also asked to
refrain from the use of anti-inflammatory drugs and all
supplements for 2 weeks prior to the downhill run and until
after the 72 h post-run visit. Compliance was assessed by a
written questionnaire that was completed at the beginning
of each visit. During Visit 2, muscle damage was induced
in all subjects by a 40-min downhill treadmill run (Gaitway
Treadmill, Kistler, Amherst, NY, USA), at -10 % grade,
at the speed corresponding to 60 % of the subjects’
VO2peak. The 40-min downhill run was a novel activity for
all subjects and was therefore expected to induce signifi-
cant DOMS. At completion of the run, subjects were given
a 3-min level cooldown walk at a self-selected pace on the
treadmill.
Cryotherapy
Ten of the subjects were randomly assigned to the cryo-
therapy treatment and ten to the control condition and were
informed of their group assignment prior to the downhill
run. Before subjects entered the tank, water temperature
was adjusted by adding ice until it reached 3–5 °C.
Immediately following this post-run assessments on Visit
2, each of the cryotherapy subjects stood quietly in the tank
of 5 ± 2 °C water that came to the top of the thigh for a
123
Eur J Appl Physiol
duration of 20 min. Water temperature was checked an
average of two times during the treatment and more ice was
added if needed. The temperature did not rise above 7 °C
during the 20-min treatment for any subject. Each of the
control subjects stood quietly in the laboratory for the same
period of time following his post-run assessments for con-
sistency with previous studies that have used no treatment as
the control condition (Eston and Peters 1999; Howatson et al.
2009; Ingram et al. 2009; Jakeman et al. 2009; Lane and
Wenger 2004; Paddon-Jones and Quigley 1997).
Swelling and soreness
Circumference of the non-dominant thigh was measured
with a tension-controlled tape measure (Creative Health
Products, Ann Arbor, MI, USA) at the midpoint of a line
drawn from the anterior superior iliac spine to the superior
pole of the patella. Thigh circumference has an intraob-
server percentage of reliability coefficient [0.98 % and
technical error of measurement averages 0.67 cm (Moreno
et al. 2003). Soreness experienced while walking down a
flight of stairs was self-reported using a VASS. The VASS
is an unmarked horizontal 100-mm line with the terminal
descriptors ‘‘no soreness’’ and ‘‘very, very sore’’. Subjects
were instructed to ‘‘think about how your legs feel’’ while
walking down the stairs and then marked their perceived
soreness on the line. Their pain score was the distance in
millimeters from the ‘‘no soreness’’ end of the line to the
subject’s mark. The intraclass correlation coefficient for a
visual analog scale has been reported to be rxx = 0.97
when used for experimental heat pain and chronic pain;
however, this study used it for soreness (Price et al. 1983).
Soreness and swelling measurements were taken prior to
the run (baseline), immediately after the cooldown, and 1,
6, 24, 48, and 72 h post-exercise.
Strength
Maximal isometric knee extensor torque of the non-domi-
nant leg was measured at 105o of knee extension (with 180o
being full extension) on a HUMAC Norm dynamometer
(CSMI, Stoughton, MA, USA) and recorded with a
BIOPAC MP150 data acquisition system (BIOPAC Sys-
tems, Inc, Goleta, CA, USA). Participants were seated with
a hip angle of 85o and pushed maximally against the
resistance arm for 3 s. The mean of the peak torque
obtained from three attempts was recorded and used for
analysis. Isometric testing of the knee extensors using this
dynamometer has been shown to have an intraclass corre-
lation coefficient of rxx = 0.95 for peak isometric torque
(LaRoche et al. 2008). Measurements were taken prior to
and immediately after the downhill run and 1, 6, 24, 48, and
72 h post-exercise.
Eur J Appl Physiol
Chemokine ligand 2
Venous blood samples from an antecubital vein were
drawn prior to and immediately after the downhill run
and 1, 6, and 24 h post-run. Two milliliters of blood was
drawn using a standard venipuncture into vacutainer tubes
containing sodium heparin. Samples were centrifuged for
15 min at 3,0009g immediately after collection. Plasma
was aliquotted and stored at -80 °C until assayed. CCL2
was quantified using human CCL2 Quantikine ELISA
kits according to the manufacturer’s instructions (R&D
Systems, Minneapolis, MN, USA). Briefly, plasma sam-
ples were incubated in a 96-well microplate pre-coated
with monoclonal antibody to human CCL2. The plate
was subsequently washed with washing buffer, leaving
only the cytokine bound to antibodies. Each sample was
then exposed to horseradish peroxidase-linked polyclonal
antibody specific for CCL2. Following another washing
step, the wells were exposed to substrate to produce a
colorimetric precipitate proportional to the amount of
CCL2 in the sample. The color reaction was terminated
and the optical density of each sample was determined at
450 nm using a microplate reader (Biotek ELx808, Bio-
Tek Instruments, Inc., Winooski, VT, USA). Cyto-
kine concentrations for the samples were determined by
comparison to a standard curve of known concentrations
of cytokines and their optical density. All samples were
run in duplicate. The intra-assay coefficient of variation
was 4.6 % and the inter-assay coefficient of variation was
3.7 %.
Statistical analyses
Estimates of skewness and kurtosis were used to screen
for normality of the data. Homogeneity of variance was
assessed using Levene’s statistic and Mauchley’s test of
sphericity. For variables that violated the assumption of
homogeneity of variances, a Greenhouse–Geisser cor-
rection factor was applied to the degrees of freedom and
subsequent corrected p values were reported in the
results. Baseline comparisons were made between groups
for descriptive statistics, CCL2, strength, thigh circum-
ference and soreness, using a one-way analysis of vari-
ance (ANOVA). To compare the differences in CCL2
between groups over time following the downhill run, a
2 9 5 (group 9 time) repeated-measures ANOVA was
used. A separate 2 9 7 (group 9 time) repeated-mea-
sures ANOVA was used to compare differences in
strength, thigh circumference, and soreness. The signifi-
cance level for all testing was set at P \ 0.05. Data are
reported as means ± SD. Effect sizes for the cryotherapy
treatment were determined from the ANOVA by partial
eta squared (g2).
Results
All data were normally distributed except reported walking
time and VASS pre-run. One subject reported that he
walked 200 min each day which is unusually high. VASS
at pre-run was skewed to the right because pre-run soreness
ratings abutted zero. All variables met the homogeneity of
variance assumption except MVC and CCL2. The Green-
house–Geisser adjusted p values are reported for these two
variables.
Subject demographics
There were no significant differences between the cryo-
therapy and control groups for demographics, reported
activity level, VO2peak, downhill running speed, or any of
the baseline measurements (Table 1). All subjects com-
pleted the 40-min run at 60 % of their VO2peak without
stopping.
Maximum voluntary contraction torque
The muscle-damaging downhill run resulted in a signifi-
cant change in maximum voluntary contraction torque
(MVC) over time (p \ 0.001, power = 1.000). Torque
decreased by an average of 6.2 % immediately following
the downhill run and this decrement persisted at 1 h post-
run. Over the next 2 days, torque recovered to pre-exer-
cise values and by 72 h post-run torque had increased
significantly above baseline values by 9.2 % (Fig. 2a).
The response to the downhill run over time explained
35 % of the variation in MVC (g2 = 0.35). There was no
significant difference in MVC between the cryotherapy
and control groups (p = 0.992, power = 0.346, g2 = 0.0).
The interaction between group and time was not signifi-
cant and explained little of the variance in MVC
(p = 0.491, power = 0.346, g2 = 0.03), leaving 62 % of
the variance unexplained.
Soreness
The downhill run elicited a significant change in soreness
over time (p \ 0.001; power = 1.00). The run caused an
average increase in VASS rating from 3.7 to 47.4 mm out
of 100 mm which remained significantly elevated above
baseline at all subsequent time points. Soreness declined at
the 6-h mark then increased again to peak at 56.4 mm 24 h
post-run and finally declined to 22.2 mm by 72 h post-run
(Fig. 2b). Sixty percent of the variation in the VASS rat-
ings can be attributed to the time at which the measures
were taken. The cryotherapy and control groups did not
differ in their VASS ratings (p = 0.256; power = 0.199),
and only 3.5 % of the variation in VASS ratings was due to
123
 Eur J Appl Physiol

Table 1 Comparison of sample

Control group Cryotherapy group p value
characteristics
Age (years) 21.5 ± 3.2 20.9 ± 0.9 0.575
Mass (kg) 75.2 ± 9.7 77.6 ± 9.9 0.589
Height (m) 1.76 ± 0.05 1.80 ± 0.04 0.075
Body mass index (kg m-2) 24.3 ± 2.5 24.1 ± 3.2 0.835
Activity level
Running distance (km week-1) 14.7 ± 12.1 18.5 ± 16.7 0.689
Walking time (min week-1) 265 ± 124 366 ± 372 0.693
Moderate intensity activity (min week-1) 249 ± 127 323 ± 271 0.441
Vigorous intensity activity (min week-1) 175 ± 165 190 ± 154 0.830
VO2peak (mL kg-1 min-1) 58.1 ± 8.1 59.7 ± 9.3 0.561
-1
Downhill running speed (m s ) 3.68 ± 0.86 3.55 ± 0.55 0.425
Downhill run distance (km) 8.83 ± 2.07 8.52 ± 1.34 0.694
Values are the mean ± SD

Thigh circumference

The downhill run did not alter thigh circumference mea-

surements over time (p = 0.151; power = 0.597;
g2 = 0.033). The cryotherapy and control groups did not
differ in thigh circumference (p = 0.677; power = 0.069;
g2 = 0.584), nor were there differences in how the groups
responded over time (p = 0.860; power = 0.170;
g2 = 0.009). For example, thigh circumference was
54.1 ± 4.2 cm post-run for the control group and
55.0 ± 3.8 cm for the cryotherapy group with no change at
24 h post-run (54.2 ± 4.4 and 54.8 ± 3.9 cm, respec-
tively), or at any other time point.

Chemokine ligand-2

Data for plasma CCL2 concentration were available for 16

Fig. 2 Peak voluntary torque and soreness over time. a Peak knee
extensor isometric torque over time. b Perceived soreness over time.
Values are the mean ± SD. *Time effect, significantly different from
pre (p \ 0.05). #Time effect, significantly different from post
(p \ 0.05)
group. Both groups showed similar responses over time
(p = 0.696; power = 0.246) with only 1.3 % of the vari-
ation being explained by the group 9 time interaction.
subjects (n = 9 cryotherapy group; n = 7 control group);
plasma from the first four subjects of the study thawed as a
result of a freezer malfunction and were discarded. The
muscle-damaging protocol had a significant effect on
CCL2 concentrations over time (p \ 0.001; power =
1.000). Average plasma CCL2 concentrations increased
from 108 to 156 pg mL-1 post-run, peaked at
251 pg mL-1 6 h post-run, and declined to 119 pg mL-1
by 24 h post-run (Fig. 3a). Fifty-nine percent of the vari-
ation in CCL2 concentrations was attributed to time, while
only 6.2 % was attributed to group and 4 % to group 9
time interactions, leaving 30 % of the variance in CCL2
unexplained (Fig. 3b). There was no significant effect of
cryotherapy on CCL2 concentrations (p = 0.116;
power = 0.344; g2 = 0.062). Groups did not differ in their
plasma CCL2 concentrations as a function of time
(p = 0.430; power = 0.217; g2 = 0.041). However, there
was a trend toward lower CCL2 concentrations in the
cryotherapy group at 6 h post-run. The peak in CCL2
concentration at 6 h post-run was 173 % higher than post-

123
Eur J Appl Physiol
Fig. 3 Plasma chemokine ligand 2 (CCL2) over time. a Comparison
of CCL2 concentration between cryotherapy and control groups over
time. b Comparison of the individual percent change of CCL2 from
the post-run (pre-treatment) time point. Values are the mean ± SD.
*Time effect, significantly different from pre (p \ 0.05). #Time
effect, significantly different from post (p \ 0.05)
run in the control group and only 146 % higher than post-
run in the cryotherapy group (control group increased from
170 to 293 pg mL-1, while the cryotherapy increased from
143 to 208 pg mL-1).
Discussion
This study explored the effects of cryotherapy on the CCL2
response to damaging exercise and contributes to previous
work on strength, swelling, and soreness measures.
Important findings include a significant effect of time for
strength, soreness, and for plasma measures of CCL2 fol-
lowing the downhill run, of which the time response for
CCL2 was previously unknown. This study demonstrated
no significant therapeutic effect of cryotherapy on any of
the variables measured, although there was a trend toward
lower CCL2 concentration following cryotherapy.
Downhill running is an ideal model for studying cryo-
therapy because it induces DOMS and an inflammatory
response. Running is a part of training for many sports, and
runners in particular tend to utilize cryotherapy (e.g., ice
baths) as a method to speed recovery. As expected, 40 min
of running down a -10 % grade at 60 % of VO2peak
induced muscle damage and inflammation. Muscle damage
was suggested by the strength decrement that did not
recover until about 48 h post-run, and the significant
DOMS which remained elevated at 72 h post-run. The
observed increases in plasma CCL2 concentrations post-
run are typical of an inflammatory response associated with
muscle-damaging exercise (Peake et al. 2005).
Strength, swelling, and soreness
Cryotherapy was not effective at attenuating the strength
decrement observed after the downhill run. None of the
variation in MVC was due to cryotherapy (0.0 %), and only
3.1 % was due to the group 9 time interaction, suggesting
that this recovery modality had little effect on reducing
strength decrement following muscle-damaging exercise.
A lack of effect of cryotherapy on strength was also
observed by other researchers (Howatson et al. 2009),
though one found a non-significant 25 % attenuation of the
strength decrement (Eston and Peters 1999). Others have
observed improvements in other performance variables
such as sprint performance (Ingram et al. 2009) and cycling
power output (Vaile et al. 2008) following cryotherapy.
Perhaps, performance of sport-specific activities is
improved with cryotherapy, but isometric strength is not,
because performing functional movements depends on the
ability to move through a normal range of motion, which is
compromised with DOMS (LaRoche and Connolly 2006).
The increase in strength seen in both groups at the 72-h
time point is likely a result of familiarization with the
isometric strength test.
Any swelling that may have occurred in the thigh as a
result of the run was not detectable with the circumference
measurement used nor was any difference resulting from
cryotherapy detectable. Most studies agree that cryotherapy
does not significantly affect swelling, specifically circum-
ference as measured with an anthropometric tape measure
(Howatson et al. 2009; Sellwood et al. 2007) or volume
measured via water displacement (Paddon-Jones and
Quigley 1997).
VASS increased dramatically in response to the run. An
interesting finding is the double peaks in soreness that
occurred in both groups immediately post-run and again at
24 h post-run. We believe the first peak represents the
acute fatigue-related pain that occurred immediately fol-
lowing exercise and the peak at 24 h post-run likely rep-
resents DOMS. At 6 h post-run, acute pain had declined
123

significantly, and possibly at a greater rate in the cryo-
therapy group (note the slopes of the lines from 1 to 6 h in
Fig. 2b), but DOMS had not yet set in causing a temporary
drop in soreness ratings. There were, however, no signifi-
cant differences between groups over time in muscle
soreness. Others who used the VASS to measure the
influence of cryotherapy on soreness have also failed to
observe any effect. For example, Jakeman et al. (2009) did
not detect a difference in soreness using the VASS at five
time points over 96 h, following ten sets of ten counter
movement jumps (Jakeman et al. 2009). Similar to our
study, Sellwood et al. (2007) observed no effect of
cryotherapy (three 1-min immersions in 5 °C water) on
soreness when rated on a 100-mm VASS at 24, 48 or 72 h
post-eccentric quadriceps exercise (Sellwood et al. 2007).
Researchers who measured muscle soreness using applied
pressure also reported no effect of cryotherapy (Eston and
Peters 1999). The results of this study do not support the
common use of cryotherapy in ameliorating soreness.
However, a few researchers have found a decrease in
soreness when cryotherapy was used during recovery. In-
gram et al. (2009) observed a significant reduction in
soreness at 24 h in those who underwent two 5-min
immersions in 10 °C water when soreness in the quadriceps
was rated on a ten-point Likert scale (Ingram et al. 2009).
Rowsell et al. (2009) also observed a reduction in soreness
at 24, 48, and 72 h throughout a soccer tournament in those
who underwent five, 1-min immersions in 10 °C water
after each match when subjects rated leg soreness on a
scale of 1–10 (Rowsell et al. 2009). One possible expla-
nation for the different results is that the coefficient of
variation for the ten-point Likert scales used in the previous
studies is lower than for the 100-mm VASS used in this
study (23.5 and 27.6 versus 65.5 %). Thus, differences in
the soreness measure used, cryotherapy protocol, exercise
performed, time of the measurements, and subject sample
confound the effects of cryotherapy on muscle soreness,
necessitating additional study.
Plasma CCL2
Plasma CCL2 concentrations followed the pattern expected
after muscle-damaging exercise with a 2–2.5-fold increase
by the 6-h post-run mark. Of the five time points at which
blood was drawn, CCL2 concentration was highest at the
6-h point. It is possible that the true peak may have
occurred before or after 6 h at a time when blood was not
drawn. This study is one of the first to quantify the time
course of CCL2 after muscle-damaging exercise and
showed a trend toward a reduction in CCL2 concentration
with cryotherapy. Peake et al. (2005) found a marked
increase in CCL2 immediately, and 1 h after downhill
running, but did not obtain plasma measures at any later
123
Eur J Appl Physiol
time points, making our study the first to measure CCL2
over a 24-h time period.
While the majority of variation in CCL2 concentration
was based on differences between subjects, cryotherapy
accounted for 6.2 % of the variation in CCL2 and the
group 9 time interaction accounted for 4.1 % of the vari-
ation. At the pre-exercise time point, the between-subject
variation in CCL2 was not unusual for plasma markers of
inflammation and muscle damage. The mean CCL2 con-
centration at this point of the study was 108 pg mL-1, with
an SD of 29 pg mL-1, which elicited a coefficient of
variation of 27.2 %. To put this in perspective, the coef-
ficient of variation for creatine kinase (a common marker
of muscle damage) at baseline in previous cryotherapy
studies was 45.1, 69.4 and 60.0 % (Howatson et al. 2009;
Ingram et al. 2009; Rowsell et al. 2009). Similar to the
highly variable changes of creatine kinase observed in
previous studies, the individual change of CCL2 after
exercise was inconsistent between individuals in both
groups in this study (Fig. 3b). Some subjects experienced
more than a 200 % increase in CCL2 from the post-run to
the 6-h time point, whereas others had minimal change.
This individual variability in the inflammatory response
contributes to the difficulty of assessing the efficacy of
cryotherapy treatment. Unfortunately, it is not possible to
definitively identify the source of the individual variation
in CCL2, but the volume, intensity, and type of previous
physical activity could modify the reaction, as could dif-
ferences in the responsiveness of individuals’ immune
systems.
Although the differences between the groups did not
reach statistical significance, CCL2 was 29 % lower in the
cryotherapy group at 6 h post-exercise. This finding
should be interpreted with caution as the response was
highly variable among subjects, especially within the
control group where one individual experienced a partic-
ularly high CCL2 peak. As this is a new area of research,
the magnitude of change in CCL2 that is clinically sig-
nificant is not yet known, but CCL2 deficiency has been
shown to impair skeletal muscle regeneration in mice
(Shireman et al. 2006). CCL2 is needed to recruit
monocytes into injured muscles to conduct phagocytosis
and produce IGF-1 for injury repair. CCL2 signaling also
up-regulates IGF-1 expression by intramuscular macro-
phages to promote skeletal muscle repair (Lu et al. 2011).
Thus, reducing CCL2 following exercise may negatively
impact adaptation. However, limiting the rise in CCL2
may be beneficial for short-term recovery, in that it may
lessen the secondary damage caused by the inflammatory
process and the associated DOMS. This could possibly
minimize performance loss in the short term, though we
did not observe an effect on maximal voluntary isometric
strength.
Eur J Appl Physiol
The study did not support the hypothesis that cryother-
apy reduces the inflammatory response to downhill running
as measured by plasma CCL2, DOMS, swelling in the
thigh, or decrement of knee extensor strength. There were
no significant differences between the groups over time for
any of the dependent variables. This raises the question of
whether or not cryotherapy is effective at reducing the
symptoms of muscle damage. The effect sizes (g2) for
group main effects and group 9 time interactions in this
study were very small for soreness, thigh circumference,
and strength, suggesting cryotherapy had almost no effect
on these measures. In fact, to detect differences, sample
sizes of 75–1,000 subjects would have been necessary for
these variables. Conversely, 21 subjects would have been
necessary to detect a significant difference in plasma CCL2
concentration, suggesting cryotherapy may have an effect
on this inflammatory cytokine. Although the current study
contained a limited number of subjects, relative differences
in CCL2 were observed due to time and treatment effects.
However, additional study is needed to more fully evaluate
the merit of cryotherapy in attenuating the inflammatory
response and hastening recovery from muscle-damaging
exercise.
Future work should clarify the effects of cryotherapy on
CCL2 and other cytokines in clinical populations, as well
as in recreational and elite athletes, with both accustomed
and unaccustomed exercises. If cryotherapy is shown to be
effective at speeding recovery, studies should determine if
there is an optimal protocol. Equally important are the
long-term effects of cryotherapy on adaptation when it is
used regularly. Because the inflammatory response is
important in mediating adaptation to exercise (Best and
Hunter 2000; Brunelli and Rovere-Querini 2008; Butter-
field et al. 2006; Chazaud et al. 2009), attenuating this
response may reduce training adaptations in those who
chronically uses cryotherapy. For example, Yamane et al.
(2006) found that cryotherapy reduced improvements in
unilateral VO2max and vascular adaptations following
training. This suggests that cryotherapy may not benefit
long-term fitness when used regularly. Perhaps, a more
effective application of cryotherapy would be multi-day
events when athletes must perform repeatedly with little
rest, and the benefits of quick recovery outweigh the risk of
attenuated long-term adaptation.
Limitations
This study used recreationally active men performing an
unaccustomed downhill running exercise, which may limit
the generalizability of the results. A number of factors were
not studied that may influence the inflammatory response
and recovery such as diet, rest, lifestyle, and additional
fitness parameters. Isometric MVC torque was used to
measure strength, but a sport-specific measure such as
sprinting time may have demonstrated greater decrements
in performance. Variability in plasma CCL2 concentration
over time necessitates a larger sample size for the attain-
ment of statistical significance between groups.
Conclusion
Forty minutes of downhill running at 60 % of VO2peak
induces muscle damage and inflammation, but the results
of the current study do not support the use of cryotherapy
as a recovery modality. Plasma CCL2 concentration, as a
marker of inflammation, increased immediately following
downhill running, peaked at 6 h post-exercise and returned
near baseline by 24 h post-exercise. Implementation of
20 min of cryotherapy at 5 °C was not effective at atten-
uating the loss of strength and increase in soreness seen
after muscle-damaging exercise, but may have mitigated
the rise in plasma CCL2 concentration.
Acknowledgments The authors would like to thank the subjects
who participated in this study. This study was conducted with no
external funding.
Conflict of interest The authors report no conflict of interest.
References
Barnett A (2006) Using recovery modalities between training sessions
in elite athletes: does it help? Sports Med 36(9):781–796
Best TM, Hunter KD (2000) Muscle injury and repair. Phys Med
Rehabil Clin N Am 11(2):251–266
Best TM, Fiebig R, Corr DT, Brickson S, Ji L (1999) Free radical
activity, antioxidant enzyme, and glutathione changes with
muscle stretch injury in rabbits. J Appl Physiol 87(1):74–82
Bleakley CM, Davison GW (2010) What is the biochemical and
physiological rationale for using cold-water immersion in sports
recovery? A systematic review. Br J Sports Med 44(3):179–187
Brickson S, Hollander J, Corr DT, Ji LL, Best TM (2001) Oxidant
production and immune response after stretch injury in skeletal
muscle. Med Sci Sports Exerc 33(12):2010–2015
Brunelli S, Rovere-Querini P (2008) The immune system and the
repair of skeletal muscle. Pharmacol Res 58(2):117–121
Buford TW, Cooke MB, Shelmadine BD, Hudson GM, Redd L,
Willoughby DS (2009) Effects of eccentric treadmill exercise on
inflammatory gene expression in human skeletal muscle. Appl
Physiol Nutr Metab 34(4):745–753
Butterfield TA, Best TM, Merrick MA (2006) The dual roles of
neutrophils and macrophages in inflammation: a critical balance
between tissue damage and repair. J Athl Train 41(4):457–465
Camus G, Deby-Dupont G, Deby C, Juchmes-Ferir A, Pincemail J,
Lamy M (1993) Inflammatory response to strenuous muscular
exercise in man. Mediators Inflamm 2(5):335–342
Chazaud B, Brigitte M, Yacoub-Youssef H, Arnold L, Gherardi R,
Sonnet C, Lafuste P, Chretien F (2009) Dual and beneficial roles
of macrophages during skeletal muscle regeneration. Exerc Sport
Sci Rev 37(1):18–22
123

Cheung K, Hume P, Maxwell L (2003) Delayed onset muscle
soreness: treatment strategies and performance factors. Sports
Med 33(2):145–164
Connolly DA, Sayers SP, McHugh MP (2003) Treatment and
prevention of delayed onset muscle soreness. J Strength Cond
Res 17(1):197–208
Eston R, Peters D (1999) Effects of cold water immersion on the
symptoms of exercise-induced muscle damage. J Sports Sci
17(3):231–238
Giraldo E, Garcia JJ, Hinchado MD, Ortega E (2009) Exercise
intensity-dependent changes in the inflammatory response in
sedentary women: role of neuroendocrine parameters in the
neutrophil phagocytic process and the pro-/anti-inflammatory
cytokine balance. NeuroImmunoModulation 16(4):237–244
Howatson G, Goodall S, van Someren KA (2009) The influence of
cold water immersions on adaptation following a single bout of
damaging exercise. Eur J Appl Physiol 105(4):615–621
Hubal MJ, Chen TC, Thompson PD, Clarkson PM (2008) Inflamma-
tory gene changes associated with the repeated-bout effect. Am J
Physiol Regul Integr Comp Physiol 294(5):R1628–R1637
Ingram J, Dawson B, Goodman C, Wallman K, Beilby J (2009) Effect
of water immersion methods on post-exercise recovery from
simulated team sport exercise. J Sci Med Sport 12(3):417–421
Jakeman JR, Macrae R, Eston R (2009) A single 10-min bout of cold-
water immersion therapy after strenuous polymetric exercise has
no beneficial effect on recovery from the symptoms of exercise-
induced muscle damage. Ergonomics 52(4):456–460
Lane KN, Wenger HA (2004) Effect of selected recovery conditions
on performance of repeated bouts of intermittent cycling
separated by 24 hours. J Strength Cond Res 18(4):855–860
Lapointe BM, Frenette J, Cote CH (2002) Lengthening contraction-
induced inflammation is linked to secondary damage but devoid
of neutrophil invasion. J Appl Physiol 92(5):1995–2004
LaRoche DP, Connolly D (2006) Effects of stretching on passive
muscle tension and response to eccentric exercise. Am J Sport
Med 34(6):1000–1007
LaRoche DP, Roy SJ, Knight CA, Dickie JL (2008) Elderly women
have blunted response to resistance training despite reduced
antagonist coactivation. Med Sci Sports Exerc 40(9):1660–1668
Lu H, Huang D, Ransohoff RM, Zhou L (2011) Acute skeletal muscle
injury: CCL2 expression by both monocytes and injured muscle
is required for repair. Faseb J 25(10):3344–55 (Epub 2011 Jun
22)
Malm C, Nyberg P, Engstrom M, Sjodin B, Lenkei R, Ekblom B,
Lundberg I (2000) Immunological changes in human skeletal
muscle and blood after eccentric exercise and multiple biopsies.
J Physiol 529(Pt 1):243–262
Malm C, Sjodin TL, Sjoberg B, Lenkei R, Renstrom P, Lundberg IE,
Ekblom B (2004) Leukocytes, cytokines, growth factors and
123
Eur J Appl Physiol
hormones in human skeletal muscle and blood after uphill or
downhill running. J Physiol 556(Pt 3):983–1000
Moreno LA, Joyanes M, Mesana MI, Gonzalez-Gross M, Gil CM,
Sarria A, Gutierrez A, Garaulet M, Perez-Prieto R, Bueno M,
Marcos A, AVENA Study Group (2003) Harmonization of
anthropometric measurements for a multicenter nutrition survey
in Spanish adolescents. Nutrition 19(6):481–486
Paddon-Jones DJ, Quigley BM (1997) Effect of cryotherapy on
muscle soreness and strength following eccentric exercise. Int J
Sports Med 18(8):588–593
Peake JM, Suzuki K, Hordem M, Wilson G, Nosaka K, Coombes JS
(2005a) Plasma cytokine changes in relation to exercise intensity
and muscle damage. Eur J Appl Physiol 95:514–521
Peake J, Nosaka K, Suzuki K (2005b) Characterization of inflamma-
tory responses to eccentric exercise in humans. Exerc Immunol
Rev 11:64–85
Price DD, McGrath PA, Rafii A, Buckingham B (1983) The
validation of visual analogue scales as ratio scale measures for
chronic and experimental pain. Pain 17(1):45–56
Rowsell GJ, Coutts AJ, Reaburn P, Hill-Haas S (2009) Effects of
cold-water immersion on physical performance between succes-
sive matches in high-performance junior male soccer players.
J Sports Sci 27(6):565–573
Sellwood KL, Brukner P, Williams D, Nicol A, Hinman R (2007) Ice-
water immersion and delayed-onset muscle soreness: a random-
ised controlled trial. Br J Sports Med 41(6):392–397
Shireman PK, Contreras-Shannon V, Ochoa O, Karia BP, Michalek
JE, McManus LM (2006) MCP-1 deficiency causes altered
inflammation with impaired skeletal muscle regeneration. J Leu-
koc Biol 81:775–785
Smith LL, McKune AJ, Semple SJ, Sibanda E, Steel H, Anderson R
(2007) Changes in serum cytokines after repeated bouts of
downhill running. Appl Physiol Nutr Metab 32(2):233–240
Tidball JG (2005) Inflammatory processes in muscle injury and
repair. Am J Physiol Regul Integr Comp Physiol 288(2):R345–
R353
Tidball JG, Wehling-Henricks M (2007) Macrophages promote
muscle membrane repair and muscle fibre growth and regener-
ation during modified muscle loading in mice in vivo. J Physiol
578(Pt 1):327–336
Vaile J, Halson S, Gill N, Dawson B (2008) Effect of hydrotherapy on
recovery from fatigue. Int J Sports Med 29(7):539–544
Yamane M, Teruya H, Nakano M, Ogai R, Ohnishi N, Kosaka M
(2006) Post-exercise leg and forearm flexor muscle cooling in
humans attenuates endurance and resistance training effects on
muscle performance and on circulatory adaptation. Eur J Appl
Physiol 96(5):572–580