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Exercise Induced Fluid Shifts are Distinct to Exercise Mode and Intensity - a
Comparison of Blood Flow Restricted and Free Flow Resistance Exercise

Article  in  Journal of Applied Physiology · April 2021

DOI: 10.1152/japplphysiol.01012.2020

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J Appl Physiol 130: 1822–1835, 2021.
First published April 29, 2021; doi:10.1152/japplphysiol.01012.2020

RESEARCH ARTICLE

Hypoxia

Exercise-induced fluid shifts are distinct to exercise mode and intensity: a

comparison of blood flow-restricted and free-flow resistance exercise
Bryan Haddock,1 Sofie K. Hansen,1,2 Ulrich Lindberg,1 Jakob Lindberg Nielsen,4 Ulrik Frandsen,4
Per Aagaard,4 Henrik B. W. Larsson,1,5 and Charlotte Suetta1,2,3
1
Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, Copenhagen University Hospital,
Copenhagen, Denmark; 2Geriatric Research Unit, Department of Geriatric and Palliative Medicine, Bispebjerg and
Frederiksberg Hospital, Copenhagen University Hospital, Copenhagen, Denmark; 3Geriatric Research Unit, Department of
Medicine Herlev and Gentofte Hospital, Copenhagen University Hospital, Copenhagen, Denmark; 4Department of Sport
Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark; and 5Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen, Denmark

Abstract
MRI can provide fundamental tools in decoding physiological stressors stimulated by training paradigms. Acute physiological
changes induced by three diverse exercise protocols known to elicit similar levels of muscle hypertrophy were evaluated using
muscle functional magnetic resonance imaging (mfMRI). The study was a cross-over study with participants (n = 10) performing
three acute unilateral knee extensor exercise protocols to failure and a work matched control exercise protocol. Participants
were scanned after each exercise protocol; 70% 1 repetition maximum (RM) (FF70); 20% 1RM (FF20); 20% 1RM with blood flow
restriction (BFR20); free-flow (FF) control work matched to BFR20 (FF20WM). Post exercise mfMRI scans were used to obtain
interleaved measures of muscle R2 (indicator of edema), R2 0 (indicator of deoxyhemoglobin), muscle cross sectional area (CSA)
blood flow, and diffusion. Both BFR20 and FF20 exercise resulted in a larger acute decrease in R2, decrease in R2 0 , and expan-
sion of the extracellular compartment with slower rates of recovery. BFR20 caused greater acute increases in muscle CSA than
FF20WM and FF70. Only BFR20 caused acute increases in intracellular volume. Postexercise muscle blood flow was higher after
FF70 and FF20 exercise than BFR20. Acute changes in mean diffusivity were similar across all exercise protocols. This study
was able to differentiate the acute physiological responses between anabolic exercise protocols. Low-load exercise protocols,
known to have relatively higher energy contributions from glycolysis at task failure, elicited a higher mfMRI response.
Noninvasive mfMRI represents a promising tool for decoding mechanisms of anabolic adaptation in muscle.
NEW & NOTEWORTHY Using muscle functional MRI (mfMRI), this study was able to differentiate the acute physiological
responses following three established hypertrophic resistance exercise strategies. Low-load exercise protocols performed to fail-
ure, with or without blood flow restriction, resulted in larger changes in R2 (i.e. greater T2-shifts) with a slow rate of return to
baseline indicative of myocellular fluid shifts. These data were cross evaluated with interleaved measures of macrovascular
blood flow, water diffusion, muscle cross sectional area (i.e. acute macroscopic muscle swelling), and intracellular water fraction
measured using MRI.

BFR; MRI; R2; mfMRI; T2

INTRODUCTION instance, has long been suspected to be a physiological stres-

Despite solid evidence that resistance training induces
muscle hypertrophy (1–3), a deeper physiological under-
standing of the underlying mechanisms is still required.
Monitoring and mapping physiological factors in vivo are
fundamental tools in identifying and decoding physiological
stressors of specific training paradigms associated with the
stimulation of muscle hypertrophy. Mechanical tension, for
sor, as resistance training with high loads is known to elicit
muscle hypertrophy in both healthy individuals and a wide
range of patient populations of all ages (4–10). In recent
years, low load resistance training performed to failure or
performed with blood flow restriction (BFR) has also gained
attention as similar hypertrophy is observed despite the low
mechanical tension (5, 7, 8, 11–15). The diversity of these
training strategies has brought focus to a larger range of

Correspondence: B. Haddock (bryan.haddock@regionh.dk).

Submitted 24 November 2020 / Revised 23 April 2021 / Accepted 25 April 2021

1822 8750-7587/21 Copyright © 2021 The Authors. Licensed under Creative Commons Attribution CC-BY 4.0. http://www.jap.org
Published by the American Physiological Society.
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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS
physiological stressors which are suspected to trigger path-
ways leading to hypertrophy which include metabolic stress
(16–18), myocellular swelling (16, 17, 19), transient hypoxia
(13, 20, 21), as well as mechanical tension (22–25).
Muscle functional magnetic resonance imaging (mfMRI)
techniques can evaluate useful parameters with regard to
the aforementioned physiological stressors. In a recent
mfMRI study, acute low load BFR exercise induced accentu-
ated R2 decreases, increases in R20 , reduced blood flow, and
similar tissue fluid mobility (diffusion) compared to a heav-
ier load in free-flow conditions (26). R2 changes of muscle
tissue during exercise, often referred to as a “T2 shift” (T2 =
1/R2), correlates with acute muscle activity (27–29) and is
metabolic in nature, as it is primarily driven by the changes
in composition, pH, and volume of intracellular fluid follow-
ing the accumulation of metabolites, such as lactate, after
loading (30–34). R2 data can also be used to estimate changes
in the relative extra-/intracellular volume of muscle tissue.
Increases in R20 (indicator of deoxyhemoglobin) combined
with blood flow measures can monitor transient hypoxia
(35–37), and diffusion imaging can detect exercise-induced
micro damage (38). Despite recent advances, however,
mfMRI has seldom been applied to study exercise protocols
known to elicit muscle hypertrophy.
The present study employed quantitative magnetic reso-
nance imaging (MRI) techniques to investigate the acute
physiological response of three differing resistance exercise
strategies which are known to induce similar levels of hyper-
trophy in longitudinal (“chronic”) settings (4–8, 10). The
three protocols encompassed high-load 70% one repetition
maximum (1RM) and low-load 20% 1RM (8, 11) in free-flow
conditions, and a low-load (20% 1RM) performed in BFR con-
dition. All of the protocols consisted of four sets of one-leg-
ged knee extension completed to contraction failure. All
mfMRI measures were acquired repeatedly to quantify both
the magnitude of exercise-induced changes but also the rate
by which they return to pre-exercise levels, the recovery rate
(k), (35, 39). Recovery rates can both be used to disentangle
the time course of physiological responses, thus reducing
the impact of the post exercise delay time of the measure-
ment but also serve as a separate quantitative parameter.
The specific aim of this study was to evaluate the exercise-
induced mfMRI response by low-load BFR exercise and low/
high load free-flow exercise to failure compared to a work
matched controlled free-flow exercise. A cross evaluation of
mfMRI results is performed using measures of macrovascu-
lar blood flow, water diffusion, muscle cross sectional area
(i.e., macroscopic muscle swelling), and an estimate of
extracellular/intracellular water fraction. It was hypothe-
sized that low-load BFR resistance exercise performed to fail-
ure would lead to amplified decreases in R2 and R20 , increases
in muscle cross sectional area (CSA) and intracellular vol-
ume, and slower recovery rates toward baseline conditions
compared to free-flow conditions.
MATERIAL AND METHODS
Participants
Ten recreationally active healthy young men volunteered
to participate in the present study. Inclusion criteria were
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recreational physical active males aged between 18 and 30
where all candidates having a known medical condition or
previous leg injury were excluded. Before entering the study,
all participants provided their written informed consent in
accordance with the Declaration of Helsinki. The experimen-
tal procedures were approved by the Regional Ethics
Committee of the Capital Region of Denmark (protocol no.
H-1-2013-146).
One week before the experimental procedures, partici-
pants had their unilateral knee extensor 5-repetition maxi-
mum (RM) load determined for the knee extensors of each
leg separately using a commercial leg extension training de-
vice (TecnoGym, Inc.). Subsequently, participants were fam-
iliarized with blood-flow restricted strength exercise by
performing two sets to failure with the right leg at 20% of
1RM. Blood-flow restricted exercise was performed using a
11-cm-wide pneumatic cuff (model: 60-7600-005, Zimmer,
Dover, OH). The occlusion cuff was placed proximally on the
thigh and inflated to a pressure corresponding to 30 mmHg
above the participants resting diastolic blood pressure meas-
ured at rest.
Exercise Protocols
The experimental procedures consisted of four different
protocols of isolated unilateral knee extensor resistance exer-
cise (Table 1) performed over two separate scanning days. In
all exercise protocols, four exercise sets were completed, af-
ter which participants were placed in the MRI scanner for ac-
quisition of data (Fig. 1). The two exercise protocols
performed on the first experimental day (day 1) were: leg A:
four sets of knee extension with 20% 1RM to task failure with
blood-flow restriction (BFR20) and leg B: four sets of knee
extension with 70% of 1RM to task failure with free-flow re-
sistance exercise (FF70).
The two exercise protocols performed on the second ex-
perimental day (day 2) were: leg A: four sets of knee exten-
sion with free-flow 20% 1RM performing the same number of
repetitions for each set as were performed during BFR20
(i.e., work-matched), on the same leg that had performed
BFR20 (FF20WM). The FF20WM protocol was introduced to
serve as a control condition to the BFR20 exercise protocol.
Leg B: four sets of knee extension with free-flow 20% 1RM
performed to task failure with the same leg that performed
FF70 on day 1 (FF20). The first and second experimental
days were separated by a minimum of 48 h. The assignment
of “leg A” or “leg B” as being the participants right or left leg
was random.
On both day 1 and day 2 the planned exercise protocols
were performed in a random order, separated by at least one
hour of passive rest. For each protocol, sets were performed
in a moderate tempo ( 3 s/repetition) separated by 45-s rest
between sets. Upon cessation of each exercise protocol, par-
ticipants were scanned to repeatedly measure a number of
MRI parameters (see Fig. 1) over a postexercise period of
20 min. In the case of the BFR20 protocol, the cuff remained
inflated throughout the four exercise sets and was released
seconds before the first post-exercise MRI measurement.
Since the scanning field of view covers both legs, scans
obtained after the second exercise session contained not
only acute data for the leg having just performed the second
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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS

Table 1. Exercise Protocols

Day 1 Day 2
Leg A BFR20 20% 1RM with blood flow restriction completed to FF20WM Free-flow 20% 1RM performing the same number
task failure each set. of repetitions for each set as were performed during
BFR20 (i.e. work matched).
Leg B FF70 Free-flow 70% 1RM performed to task failure each set. FF20 Free-flow 20% 1RM performed to task failure each set.
All participants performed four separate unilateral knee extensor resistance exercise protocols in total, two on each scanning day (day
1 and day 2). Each protocol consisted of four sets of exercise performed at a moderate tempo and with 45-s rest between sets where par-
ticipants were placed in the MRI scanner immediately after the fourth set. The assignment of the right/left leg to be “leg A” or “leg B”
was randomized as was the order exercise protocols performed on a given scanning day. The BFR20, FF70, and FF20 exercise protocols
have previously been documented to elicit similar degrees of hypertrophy over longer training periods as opposed to FF20WM which
served a control condition. BFR20, 20% 1RM with blood flow restriction; FF, free flow; FF20, free flow 20% 1RM; FF20WM, free-flow con-
trol work matched to BFR20; FF70, free flow 70% 1RM; RM, repetition maximum; R2 0 , indicator of deoxyhemoglobin; T1/2, half-life.

exercise protocol but also late timepoints (80 min) from the
leg having performed the first exercise protocol of the day as
well. As the order of the two exercise protocols performed
each day was randomized, late time points were obtained for
five of the 10 participants for each protocol.
MRI Sequences
Diffusion, R2 and R2 data were acquired over the same
field of view (475  215 mm2) covering a transaxial slice of
the legs at mid femur and included SPAIR fat suppression on
a Philips 3 T Achieva dStream scanner using a 16-channel an-
terior coil strapped over the thighs of both legs. Axial scans
were performed at 50% femur length (mid-thigh) as identi-
fied from a frontal scout scan covering the entire femur.
After each exercise paradigm, the placement of the coil and
participant was controlled to avoid displacement.
Diffusion weighted (DWI) data were acquired for 13 b-val-
ues (0, 100, 200, 250, 300, 350, 400, 450, 550, 700, 850,
1,000, 1,300 s/mm2) and six directions for three 5-mm slices
using a TR/TE of 1,100 ms/98 ms. Data were acquired in a
matrix of 160  69 with an EPI factor of 69 reconstructed to a
256 matrix with reconstructed voxels of 1.86  1.86 mm2.
Maps of the mean apparent diffusion (MD) and fractional an-
isotropy (FA) were calculated from a monoexponential fit to
the diffusion images comprising of b-values from 200 to
800 s/mm2 using the Diffusion Toolbox version 3.0 from FSL
(40). Data from the b-values above 800 were acquired for an
analysis outside the scope of the present study.

R2’

Figure 1. Experimental protocol. On each experiment day, participants performed two separate exercise protocols, one with each leg, in a randomized
order. Both scanning days began with a baseline scan where all measures were acquired once. After baseline, participants performed the first leg's uni-
lateral knee extensor protocol for the day and then underwent a 20-min MRI scan with interleaved measures of R2 0 , R2, flow (PC), and diffusion (DWI).
After scanning, participants rested for 40 min, and then performed the second leg's exercise protocol of the day on the opposite leg followed by a sec-
ond post exercise MRI scan. The scanning field of view located the mid-thigh axial region and comprised both legs centered, to allow both acute meas-
ures of the exercised leg and late measures of the opposite leg to be included in the second scan session. The scanning procedure on the second
experimental day was identical to the first, except that BFR20 and FF70 protocols were substituted with FF20WM, and FF20 protocols, respectively.
BFR20, 20% 1RM with blood flow restriction; DWI, diffusion weighted imaging; FF, free flow; FF20, free flow 20% 1RM; FF20WM, free-flow control work
matched to BFR20; FF70, free flow 70% 1RM; PC, phase contrast; RM, repetition maximum; R2, indicator of edema; R2 0 , indicator of deoxyhemoglobin.

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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS
R2 data were acquired using a turbo spin echo sequence
(TSE factor 20) with TR 1,500 ms and 20 echo times (TE = n 
4.9 ms). One 5-mm slice was acquired with a 68  160 matrix
and reconstructed to a 256 recon matrix with a pixel size of
1.75  1.75 mm2 having a sense factor of 1.5 and a scan time
of 70 s.
A monoexponential R2 map was calculated as a least
squares minimization of:
Si ¼ S0  eTEi R2 þ C
where Si is the MRI signal as a function of echo time (TE),
So the unattenuated signal, and C is a constant that
includes signal from spins with a range of relaxation rates
much slower than that of the majority of spins in the tissue
[R2(C)<< R2(tissue)] with limited attenuation in the given
range of TE values applied in the present experiments.
The fraction of the total signal that appears constant, C/
(C þ S0), was used as an estimate of the fraction of water in
the extracellular compartment (EC) of muscle tissue (34), and
(1-EC) · CSA was used as an estimate of intracellular volume.
CSA/R2 data were acquired using a gradient echo
sequence with 10 echoes TE1/DTE 8.0 ms/7.4 ms TR 266 ms,
a flip angle of 40 degrees, and a fast field echo readout with
an EPI factor of 5. Four dynamics of three 5-mm slices cover-
ing both legs at the mid femur with a field of view of 475 
215 mm2 reconstructed to a 256  256 recon matrix with a
pixel size of 1.85  1.85 mm2. SPAIR fat suppression was
applied. CSA was determined by drawing a single region of
interest around the quadriceps muscle.
The total rate of dephasing (R2) was calculated by least
squares minimization of the equation:

Si ¼ S0  eTEi R2 þ C
from which R’2 could be calculated as the difference between
R2 and R2:
0
R2 ¼ R2  R2
Muscle blood flow was calculated using phase contrast MRI
(PC-MRI) with 100 cm·s1 velocity encoding and an acquisi-
tion time of 10 s as previously described (26). A single 6-mm
slice with a 256  132 acquisition matrix reconstructed to
voxel dimensions of 1.6 mm  1.6 mm was acquired using a
TR/TE of 10.7/6.5 ms and turbo field echo factor 50.
Macrovascular arterial and venous vessels within the quadri-
ceps muscles were identified using k means clustering (5
bins and unrestricted cluster size using velocity maps and
magnitude images as input and an adaptive threshold of 10
percent above average absolute water velocity of the quadri-
ceps; Refs. 26, 41, and 42).
To characterize the rate of return to baseline levels follow-
ing the acute exercise intervention, absolute changes in post
exercise values for R2, R20 , CSA, MD, FA, and blood flow were
fitted to a function for exponential decay functions:
QðtÞ ¼ Qpost  ek ðtTDpost Þ
with t denoting time from exercise stop in minutes, Q(t) the
absolute difference for the given parameter from baseline as
a function of t, Qpost the absolute difference from baseline of
the first post exercise measurement and TDpost the time
delay from exercise stop until post exercise values begin to
decay toward baseline (TDpost limited to a minimum t 
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TDpost = 0). The parameter k denotes an exponential rate
constant (units·min1) reflecting the rate at which post exer-
cise parametric values return to their baseline level following
cessation of the exercise protocol. The rate of recovery is also
expressed by its half-life, T1/2 = ln(2)/k, which is the time in
minutes required for the difference between the post exer-
cise parametric value and baseline value to be reduced by
half.
Statistical Analysis
Sample size was calculated based on the primary end
point of R2 changes in percent. A sample of eight partici-
pants was found necessary to detect a difference of 5 per-
centage points in R2 changes between exercise protocols
with >98% certainty based on a R2 measurement uncertainty
of 3%.
Values for R2, R20 , FA, and MD of the quadriceps muscle
were obtained from regions of interest in the vastus lateralis,
vastus intermedius, vastus medialis, and rectus femoris
muscles. A mixed-effects model was used to test for differen-
ces of measured changes in quadriceps R2, muscle CSA, FA,
MD, and blood flow at the time of first post exercise measure
between the four exercise protocols with participants set as a
random effect. The first analysis tested for a significant effect
of exercise protocol on the aforementioned variables (fixed
effect = exercise protocol performed, dependent variable =
change from baseline to first post exercise measure in varia-
bles’ ROI values, random effect = participant). A second
mixed model analysis was performed to compare the same
variables for differences between day 1 and day 2 measures
and right versus left leg to identify long-term effects from
exercise protocols (fixed effect = leg A or leg B, dependent
variable = difference in parameter's baseline measure
between day 2 and day 1, random effect = participant). A
third mixed model analysis was performed to test for correla-
tions between each of the acute mfMRI—R2 and R20 —changes
after exercise with changes in CSA, blood flow, EC, and MD.
Significant correlates identified from the third mixed model
analysis were entered into a linear regression model where
goodness of fit was evaluated with a Pearson’s adjusted R2
value. Linear regressions having an adjusted R2 value above
0.2 and a P value of less than 0.05 were considered minimal
criteria for reporting correlations. Image coregistration, ROI
analysis, calculations, and statistical analysis were per-
formed with software created in MATLAB 2016 b
(MathWorks, Natick, MA). Unless stated otherwise, values
are presented as group mean ± SD. Data points in figures rep-
resent group mean values with error bars indicating stand-
ard error of the mean.
RESULTS
All recruited participants (age 24 ± 3 yr, exercise/week 7.4 ±
4.2 h) completed the four exercise protocols and MRI scans. All
baseline measurements and absolute changes induced by the
exercise protocols are presented in Table 2. The average 1RM for
leg A (BFR20 and FF20WM) and leg B (FF70 and FF20) was
34 ± 7 and 35 ± 6 kg. Participants’ right leg had a higher 1RM
(0.8 ± 0.3 kg, P = 0.023), CSA (3.0 ± 0.4 cm2, P = 0.013), and both
lower R2 (1.1 ± 0.2 s1, P = 0.035) and FA (0.04 ± 0.01, P = 0.014)
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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS

Table 2. MRI parameters obtained pre and post BFR and free-flow muscle exercise
Absolute Change in Value Due to Exercise (First Measure post Exercise minus Baseline)
Parameter Baseline BFR20 FF70 FF20 FF20WM
1
R2, s 29.0 ± 0.5 4.0 ± 1.5 2.9 ± 2.1 4.2 ± 1.8 3.4 ± 1.6
(P = 0.0103) (n.s) (P = 0.0182)
1
R2 0 , s 13.5 ± 3.3 5.9 ± 4.2 5.4 ± 4.1 7.7 ± 3.6 5.6 ± 2.3
(n.s) (n.s) (P = 0.0273)
CSA, cm2 77.6 ± 13.4 10.2 ± 3.1 4.7 ± 4.8 6.7 ± 4.3 6.4 ± 3.6
(p = 0.0001) (n.s) (n.s)
Venous flow, mL/min 43.1 ± 34.8 79.0 ± 49.7 117.8 ± 59.2 119.3 ± 60.6 98.1 ± 55.7
(n.s) (n.s) (n.s)
Arterial flow, mL/min 25.0 ± 23.3 96.9 ± 46.2 123.4 ± 59.9 94.3 ± 43.5 130 ± 57.3
(n.s) (n.s) (n.s)
2
MD, mm /s 1.2 ± 0.1 0.4 ± 0.2 0.3 ± 0.1 0.3 ± 0.1 0.3 ± 0.1
(n.s) (n.s) (n.s)
FA 0.4 ± 0.09 0.1 ± 0.09 0.1 ± 0.06 0.09 ± 0.06 0.09 ± 0.07
(n.s) (n.s) (n.s)
Recovery rates
kR2, min1 0.10 ± 0.06 0.13 ± 0.07 0.10 ± 0.04 0.13 ± 0.07
(P = 0.0043) (n.s) (P = 0.0033)
1
kR2', min 0.07 ± 0.10 0.08 ± 0.09 0.05 ± 0.05 0.11 ± 0.12
(P = 0.0028) (P = 0.0199) (P = 0.0003)
kCSA, min1 0.08 ± 0.09 0.08 ± 0.10 0.10 ± 0.12 0.05 ± 0.03
(n.s) (n.s) (n.s)
1
kFlow, min 0.16 ± 0.12 0.12 ± 0.06 0.13 ± 0.08 0.11 ± 0.06
(n.s) (n.s) (n.s)
kMD, min1 0.012 ± 0.003 0.013 ± 0.004 0.012 ± 0.002 0.014 ± 0.003
(n.s) (n.s) (n.s)
Values presented are mean values from quadriceps ROIs ± SD. For the recovery rates of macrovascular blood flow, the mean of partici-
pants arterial and venous blood flow were used to yield a combined recovery rate. Baseline values are averaged from the baseline meas-
ures of both scanning days. P values represent significant differences between the exercise protocols known to induce hypertrophy and
the “control” exercise protocol FF20WM where “n.s” denotes not significant (P > 0.05). BFR20, 20% 1RM with blood flow restriction; CSA,
cross sectional area; MD, mean apparent diffusion; FA, fractional anisotropy; FF, free flow; FF20, free flow 20% 1RM; FF20WM, free-flow
control work matched to BFR20; FF70, free flow 70% 1RM; RM, repetition maximum; ROI, region of interest; R2, indicator of edema; R2 0 ,
indicator of deoxyhemoglobin.

baseline values than the left leg. Due to right-left randomization,
however, no significant differences between baseline measures
of the participants’ leg A and leg B at the first experimental day
were observed. Average elapsed time between the first and sec-
ond experimental day was 7 ± 3 days. Time between cessation of
exercise to the first measure time point was 3.3 ± 0.5 min for
muscle blood-flow, 3.5 ± 0.5 min for CSA, 3.8 ± 0.5 min for
R2, and 6.9 ± 0.5 min for diffusion. The mean exercise vol-
ume (load  repetitions) for FF20 was 850 ± 242 (7 ± 1 kg
lifted a total of 120 ± 6 repetitions during the four sets) and
840 ± 154 (25 ± 4 kg  34 ± 5 repetitions) for FF70. With
BFR20 the exercise volume was significantly lower (P =
0.003) with a mean exercise volume of 644 ± 158 (7 ± 1 kg 
95 ± 5 repetitions) during BFR20. The same load and repe-
titions performed under BFR20 were repeated under free-
flow conditions during the FF20WM protocol. Average cuff
pressure during the BFR protocol was 102 ± 5 mmHg.
R2
Averaged across all exercise protocols, R2 decreased by
3.6 ± 1.8 s1 after exercise which corresponds to a relative
decrease of 12.4% from baseline (Table 2, Fig. 2). Mean recov-
ery rate for R2 was 0.11 ± 0.07 min1 corresponding to a halflife
(T1/2) of 6.1 min. Changes in R2 were protocol dependent (P =
0.001) with almost identical post exercise reductions for
BFR20 and FF20 (4.0 ± 1.5 s1 and 4.2 ± 1.8 s1) exceeding
R2 reductions after FF20WM and FF70 by 30% (Table 2, Fig.
3). Rate of recovery was slower for BFR20 and FF20 compared
to FF20WM with a similar difference in T1/2 of 2.3 min between
them and the control protocol. FF70 had the same rate of re-
covery as FF20WM. The same differentiation was observed for
extracellular volume fraction, EC, (fraction of muscle water
having low R2 values). A higher fraction of total extracellular
muscle water volume was observed after the BFR20 and FF20
exercise protocols (16.4 ± 0.7% and 16.6 ± 0.8%) than FF20WM
and FF70 (15.8 ± 0.7% and 15.7 ± 0.7%; Fig. 4). A significant
group right-left leg discrepancy was observed where mean R2
decreases in the exercised right leg exceeded those of the left
leg by 1.2 ± 0.3 s1 across protocols (P < 0.001). At baseline,
the extracellular water fraction was 10.6 ± 2.9% with no signifi-
cant differences between participants’ legs.
Mixed effects modeling indicated absolute changes in R2
(s1) to be dependent on changes in muscle diffusion, MD
(mm2/s), and EC (%). Linear regression analysis using these
parameters corrected for participant and right versus left leg
yielded the following equation:
DR2 = 1.7·DMD  0.37·DEC  0.4 (P < 0.001)
The goodness of fit values were R2 = 0.79 and adjusted
R2 = 0.70.
Muscle Cross Sectional Area
Baseline quadriceps muscle CSA was 77.6 ± 13.4 cm2 which
increased across exercise protocols by an average of 9% or

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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS
7.0 ± 4.4 cm2 (Table 2). Following BFR20 Quadriceps muscle
CSA increased by 10.2 ± 3.1 cm2 corresponding to 13.1% gain,
which was significantly more than the other exercise proto-
cols (P = 0.01; Fig. 4). Acute changes in muscle CSA were low-
est for FF70 with changes of 4.7 ± 4.8 cm2. Collectively, acute
increases in CSA remained higher throughout the first
20 minutes of post exercise recovery when participants per-
formed BFR20, and lower after FF70, exercise than the con-
trol FF20WM protocol (P < 0.001 and P = 0.023, respectively).
Average recovery rate in muscle CSA was 0.08 ± 0.09 min1
which corresponds to an average T1/2 of 9 min. There were no
significant differences in CSA recovery rate between exercise
protocols.
Upon cuff release after BFR20 exercise, a rapid decrease in
CSA was noted where dilated veins were observed to decrease
in circumference. This post release effect typically was nor-
malized within the initial 30 s of the MRI scan for R2/CSA
data (the first post exercise measure) and were not observed
in the following interleaved MRI measures. Post exercise
changes in intracellular volume only reached statistical signif-
icance following BFR20 exercise (2.3 ± 1.4%, P = 0.018; Fig. 4).
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Figure 2. Maps of changes in R2 and R2 0
values after exercise protocols. Examples
are given for the changes in R2 from base-
line to the first postexercise measure (A)
and similarly for R2 0 (B) for a representa-
tive participant. On the first day, this partic-
ipant performed FF70 first and then, after
scanning and a rest, performed BFR20
exercise with the other leg. Note that at
the time of the second post exercise scan,
R2 and R2 0 values in leg B quadriceps
have almost returned to baseline from the
first training. On the second day the partic-
ipant performed FF20 exercise first on the
same leg as FF70 (leg B) and then
FF20WM on the same leg that performed
BFR20 (leg A) after having rested. The
right-left assignment of legs to be leg A or
B was randomized as was whether leg A
or leg B exercised first. Thus, late time-
points in the second scans were acquired
for FF70 and FF20 (as is the case for this
participant) in the participants (n = 5) hav-
ing performed these exercises first, and
for BFR20 and FF20WM in the remaining
participants (n = 5). BFR20, 20% 1RM with
blood flow restriction; FF, free flow; FF20,
free flow 20% 1RM; FF20WM, free-flow
control work matched to BFR20; FF70,
free flow 70% 1RM; RM, repetition maxi-
mum; R2, indicator of edema; R2 0 , indicator
of deoxyhemoglobin; D, change.
Vascular Blood Flow
Post exercise macrovascular venous flow and arterial flow
did not differ significantly between exercise protocols. Large
interindividual variations were noted for both venous and
arterial flow (Table 2, Fig. 5). Intraindividual variations in
flow depending on the exercise protocol performed were
considerably smaller. The average recovery rate of venous
and arterial blood flow combined was 0.13 ± 0.09 min1 cor-
responding to a T1/2 of 5.4 min.
Diffusion
Baseline MD and FA values were 1.2 ± 0.1 mm2/s and
0.4 ± 0.09, respectively. MD values increased on average by
0.33 ± 0.1 mm2/s after performing exercise corresponding to
an increase of 27.5% (Fig. 6). There were no significant differ-
ences between exercise protocols in the post exercise
increases and decreases in MD and FA, respectively. The av-
erage recovery rate of MD determined from 5 min and 25 min
after exercise was 0.013 ± 0.03 min1 which corresponds to a
T1/2 of 54 min.
1827
 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS

Figure 3. Recovery of post exercise R2 values toward baseline. A: mean relative post exercise changes from baseline (mean ± SE) in knee extensor R2
are presented for all four exercise protocols. In this timeframe, changes induced by BFR20 and FF20 differed from that of FF20WM and FF70 (P < 0.001)
with no significant differences within these subgroups. Initially, post exercise (3.9 min) R2 changes were 0.92 s1 greater for FF20 than FF20WM (P =
0.035). Recovery rate coefficients (kR2) for quadriceps ROIs are presented in B with BFR20 and FF20 demonstrating a slower recovery rate than
FF20WM and FF70 (P < 0.001). BFR20, 20% 1RM with blood flow restriction; FF, free flow; FF20, free flow 20% 1RM; FF20WM, free-flow control work
matched to BFR20; FF70, free flow 70% 1RM; RM, repetition maximum; R2, indicator of edema.

R20 resistance exercise protocols to failure (FF20 and BFR20) dif-

Mean recovery rate for R20 was 0.068 min1 corresponding
to a half-life (time for the difference from baseline to be
reduced by one half) of 10.2 min. FF20 decreased more than
FF20WM (P = 0.0273) and all three protocols (FF20, BFR20,
and FF70) had a lower rate of recovery than FF20WM (Table
2, Fig. 7). There were no significant right versus left leg differ-
ences. Mixed-effects model fitting revealed absolute changes
in R20 (s1) to be dependent on changes in muscle macrovas-
cular blood flow (Vflow, mL/min), CSA (%), and EC (%). A lin-
ear regression using these parameters gave the following
equation with a goodness of fit of R2 = 0.69 (adjusted
R2 = 0.51).
DR20 = 0.26 DCSA  0.017 DVflow  0.86 DEC  5.9(P < 0.001)
Differences between Day 1 and Day 2
On the second scanning day (day 2) both legs demon-
strated lower mean baseline R2 (reduced by 0.5 s1 ± 0.22;
P = 0.034) and higher mean baseline MD (elevated by
0.05 ± 0.024 mm2·s1 (P = 0.030). By separating diffusion
into directions along the longitudinal axis of the muscle fiber
and radial to the muscle fibers cross section, the increase in
radial diffusion, 0.046 ± 0.025 mm2/s (P = 0.048) reached sta-
tistical significance.
DISCUSSION
Our main finding was that the present MR-based imaging
protocols were able to differentiate the exercise-induced
mfMRI response of these fundamental diverse resistance
exercise protocols. Low load type of resistance training per-
formed to failure either with blood flow restriction (BFR20)
or without (FF20) elicited a higher mfMRI response. The
acute mfMRI measures include changes in muscle R2 (indi-
cator of metabolically driven fluid shifts), R20 (indicator of
deoxyhemoglobin concentrations), thigh cross sectional area
as well as muscle blood flow and diffusion. Both low load
fered from the low load work matched control (FF20WM)
which wasn't performed to task failure and is known to
induce little hypertrophy. Interestingly, the high load to fail-
ure protocol, FF70, also induced smaller R2 and R20
reductions, smaller acute increases in CSA and smaller
extracellular increases compared to the low load BFR20 and
FF20 protocols. Moreover, mfMRI measures obtained exhib-
ited longer recovery periods after BFR20 and FF20 compared
to the heavy type FF70 resistance exercise. BFR20 differed
from all free-flow exercise protocols in that it induced larger
acute gains in muscle CSA, which included significant
increase in intracellular volume as well as extracellular vol-
ume. Furthermore, the intracellular volume increase
induced by BFR was sustained over a longer post exercise pe-
riod. The effect of BFR versus free-flow training was not the
same for all participants. Notably, the observed changes in
R2 and R20 values in the BFR condition compared to the free-
flow conditions to failure (FF20, FF20WM and FF70) varied
greatly between individuals.
How Measured MRI Parameters Differentiate the
Exercise Protocols
The present study confirms our hypothesis that BFR exer-
cise induces larger acute increases in muscle CSA and esti-
mated intracellular volume than free-flow protocols. With
regard to the remaining MRI parameters, BFR either induced
similar changes as FF20 (R2, R20 , and EC) during the studied
time period or there was no differentiation between exercise
protocols at all (MD, FA, and blood flow). Despite a differ-
ence in exercise volume, BFR20 and FF20 induced similar
changes in R2, R20 and identical acute shifts in the extravascu-
lar fraction of muscle volume. Likewise, FF70 and FF20WM
induced a similar attenuated acute response in these param-
eters despite FF70 involving larger exercise loads, and
greater exercise volume since being performed to task failure
as opposed to FF20WM. The calculation of recovery rates
revealed a similar trend between the different exercise proto-
cols, where accentuated decreases in R2 and R20 values

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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS

Figure 4. Post exercise changes in muscle cross sectional area and extracellular space volume fraction. Post exercise knee extensor CSA (A) was
increased more after BFR20 (after cuff release 3.5 min post exercise) than the other exercise protocols (P < 0.001). As the order of the two exercise
protocols performed on a given day was randomized, each exercise protocol was allocated as the first exercise of the day in five out of 10 participants.
Late timepoints of the first exercised leg obtained from the second scan (n = 5 each) are included to indicate progression. After 80 min, acute muscle
CSA gains induced by BFR20 remained elevated above baseline (P = 0.02), but all other quadriceps muscles having performed free-flow protocols
were below baseline (P < 0.001). After exercise ( 3.8 min post exercise), the fraction of extracellular muscle water (B) increased for all protocols and af-
ter 80 min, the estimated extracellular volume fraction was lower than baseline values regardless of the exercise protocol (P < 0.001). Post exercise
changes in intracellular volume (C) only reached statistical significance following BFR20 exercise. Since the first CSA measure precedes the first EC
measure, the first time point for the estimate of intracellular volume changes was not included for BFR20, as it would be affected by the rapid changes in
blood volume occurring immediately after cuff release. BFR20, 20% 1RM with blood flow restriction; CSA, cross-sectional area; FF, free flow; FF20, free
flow 20% 1RM; FF20WM, free-flow control work matched to BFR20; FF70, free flow 70% 1RM; RM, repetition maximum.

induced by BFR20 and FF20 returned to baseline at a slower
rate than FF70 and FF20WM. Notably, similar exercise vol-
umes were reached during the high and low load free-flow
exercise protocols (FF70 and FF20) performed to contraction
failure. The application of BFR (BFR20) reduced the exercise
volume to contraction failure significantly (Table 1). In con-
trast, diffusion data, MD and FA, did not reveal any signifi-
cant post exercise differences between exercise protocols
suggesting that overall mobility in the targeted muscle tissue
water is similar during this time frame regardless of the exer-
cise paradigm performed. Likewise, macrovascular arterial
and venous blood flow during the studied post exercise time
period did not differ significantly between exercise proto-
cols. The similarity of these measured parameters indicate
that even though the overall movement of fluid, both in
blood flow and diffusion within the tissue, is elevated follow-
ing exercise, the magnitude and rate of normalisation is not
sensitive to the differences between the exercise protocols
studied in this time frame. This indicates that the observed
differences in post exercise R2, R20 , CSA, EC, and intracellular
volume changes between protocols are not driven by post
exercise blood flow and fluid mobility. In a recent acute
fMRI study (Ref. 26), we examined exercise protocols differ-
ing in exercise loading (low versus high) and BFR versus
free-flow conditions, comparable to the conditions of the
present study. Although all exercise was performed inside
the MR scanner, no significant differences were observed
between exercise protocols in terms of diffusion and blood
flow 3 min after exercise stop, respectively, despite obser-
vations that macrovascular flow was elevated by BFR or
high-load free-flow conditions within the first minute follow-
ing exercise stop (26). Collectively, these and the present
observations indicate that the observed differences between
exercise protocols in post exercise R2, R20 , CSA, EC, and intra-
cellular volume changes are not determined by differential
changes in post exercise flow and fluid mobility. Again in
our preceding study (Ref. 31), even the elevated hyperemia
following the release of the cuff after BFR had little effect on
R2 contrary to the decreases it provoked in R20 . The first post
exercise measures of R20 changes and venous flow measures

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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS

Figure 5. Macrovascular venous and arte-

rial flow. Post exercise macrovascular ve-
nous flow (A) and arterial flow (B) did not
differ between exercise protocols. Large
interindividual variations were noted for
both venous and arterial flow (bottom).
Intraindividual variations in flow induced by
the four exercise protocols were consider-
ably smaller. In the bottom, flow measures
for the four exercise protocols of each indi-
vidual (connected by a vertical line) is plot-
ted against each participant’s mean flow.
BFR20, 20% 1RM with blood flow restriction;
FF, free flow; FF20, free flow 20% 1RM;
FF20WM, free-flow control work matched to
BFR20; FF70, free flow 70% 1RM; RM, repe-
tition maximum.

were found to have a weak correlation across all protocols as
did changes in R2 and MD values post exercise. During the
post exercise time period (>3 min), recovery to baseline con-
ditions occurred quite independently of one another as MD
returned to baseline at a much slower rate than R2 values
and venous flow returned to baseline at a faster rate than R20
values. It is important to note that in this post exercise time
frame, R20 values contain differing information compared to
during exercise where R20 increases have been verified to cor-
relate with deoxygenated hemoglobin levels (36). This study
confirms a post exercise increase in R20 which could indicate
higher venous blood oxygenation than during rest
conditions.
Using mfMRI to Assess Hypertrophic Potential of
Exercise Protocol
It has been widely argued that muscle hypertrophy is
stimulated by two primary mechanisms: mechanical tension
and metabolic stress (6, 9, 16, 17, 24). Although no one pa-
rameter measured in the present study differentiated the
FF70 protocol—with higher tension—from the low load pro-
tocols, some metabolically driven observations were made.
The amplified R2 decreases observed after the BFR20 and
FF20 exercise protocol support our initial hypothesis that R2
changes would be sensitive to the elevated levels of meta-
bolic stress associated with these exercise strategies as
opposed to load, exercise volume or total metabolic activity.
Metabolic stress levels are often evaluated from increased
myocellular lactate concentrations, reduced PCr concen-
trations and lower pH. Shifts in R2 have also been reported to
correlate with metabolic markers in muscle tissue such as
glucose uptake (28), decreases in phosphocreatine concen-
trations (43), and pH (32). During resistance exercise, a
higher exercise load (>70% 1RM) requires more ATP hydro-
lyzed per contraction and can produce faster changes in PCr,
lactate and [H þ ] concentrations than lifting a lower load.
When exercising to fatigue, however, end point metabolic
stress tends to be higher after resistance training with lower
loads (<35% 1RM) (44–49) than high loads. The use of lower
relative training loads allows for more repetitions to be per-
formed before reaching contraction failure, hence resulting
in more sustained metabolic stress (due to more pronounced
glycolysis) in a larger part of the active myofibers at the point
of contraction failure (50). In the present study BFR20 and
FF20 exercise to failure were employed to represent proto-
cols with higher metabolic stress and were therefore pre-
dicted to cause larger R2 decreases than FF20WM and FF70.
This suggests that metabolic stress is a primary factor driv-
ing acute R2 changes, given that anaerobic energy systems
are reported to be more strongly involved in the BFR exercise
conditions (BFR20) compared to matched free-flow condi-
tions (FF20wm) (51, 52) and to be more heavily taxed in the
low-load (FF20) compared to high-load (FF70) free-flow exer-
cise performed to failure (44, 45). Fleckenstein et al. (27)
were the first to report that changes in R2 reach a plateau
with continued repetitions and that although the rate at
which R2 changed with increasing contractions was load de-
pendent, the point at which R2 decreases plateaued was not

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 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS
load dependent. Jenner et al. (29) later confirmed that the
plateau is not determined by a maximal limit to R2 changes,
but instead reflects a steady state determined by the exercise
intensity independent of the load or total work performed.
Even after repeated sets of exercise, measured R2 decreases
have been shown to reflect the same heterogeneous distribu-
tion as relative muscle glucose uptake during exercise (28).
Recently, R2 changes have been demonstrated to quickly pla-
teau in the first of four sets of conventional free-flow resist-
ance exercise to failure establishing a new steady state that
was maintained throughout the remaining exercise sets (26).
In the same study, it was observed that although CSA
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Figure 6. Mean diffusion (MD) and frac-
tional anisotropy (FA) in the knee extensor
and flexor muscles at baseline and follow-
ing acute exercise. Exercise induced
changes in MD (A and B) and FA (C and D)
values in all muscle groups (P < 0.001) but
did not vary between the four exercise
protocols. During 20 min of rest, no differ-
ences in recovery rate was observed
across exercise protocols as MD values
decreased (P = 0.001) and FA values
increased (P = 0.001) toward baseline.
Changes in MD and FA for hamstring
muscles, antagonists to the knee exten-
sion exercises performed, were reciprocal
to those of the quadriceps although of a
lower magnitude. BFR20, 20% 1RM with
blood flow restriction; FF, free flow; FF20,
free flow 20% 1RM; FF20WM, free-flow con-
trol work matched to BFR20; FF70, free
flow 70% 1RM; RM, repetition maximum.
increases and R2 decreases already reached a steady state af-
ter the first of four sets for free-flow exercise to failure, R2
values continued to decrease and muscle CSA increased over
the subsequent sets of BFR exercise (26). This would appear
to result from the effect BFR has on muscle tissues ability to
meet metabolic demands as venous occlusion alone, without
exercise, induces only minor R2 changes that recover quickly
(36, 37, 53). In the present study, not only were acute R2
decreases accentuated by BFR, but the recovery rate to pre-
exercise levels was also slower than observed in the FF20WM
protocol. Our findings match those of Fisher et al. (53) who
reported that R2 recovery rates were not determined by the
Figure 7. A and B: recovery of post exer-
cise R2 0 values toward baseline. Post
exercise R2 0 values decreased more after
the FF20 exercise protocol than FF20WM
(P = 0.0273) and all hypertrophic protocols
(FF20, BFR20, and FF70) had a lower rate
of recovery than FF20WM. BFR20, 20%
1RM with blood flow restriction; FF, free
flow; FF20, free flow 20% 1RM; FF20WM,
free-flow control work matched to BFR20;
FF70, free flow 70% 1RM; RM, repetition
maximum; R2 0 , indicator of deoxyhemo-
globin; T1/2, half-life.
1831
 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS
load applied during resistance exercise and did not observe a
significant correlation between acute exercise-induced
increases in CSA and changes in R2.
Interpretation of R2, EC, and Intracellular Volume Changes
The observed relationship between CSA and R2 in the
present study challenges simplistic models regarding how
exercise related factors, such as cell swelling, drive R2
changes. The intracellular buildup of several metabolites in
response to intensive regimes of exercise loading is in itself
believed to be a primary factor creating an elevated osmotic
gradient thought to drive the intracellular water retention
observed by Armstrong (54). The metabolically driven intra-
cellular water retention contributes to both acute R2
decreases and CSA increases interlinking the two measures.
Baseline CSA values were similar to previous reports of
77.5 ± 3.0 cm2 for young men (55) and the observed post exer-
cise increases in CSA corresponded closely to the acute post
exercise increases of 8%–10% reported previously (56). The
more pronounced increases observed in post exercise CSA
with BFR compared to free-flow resistance exercise also
aligns with previous observations (11) with acute increases
after BFR exercise exceeding that of free-flow exercise by
50%. However, despite that the CSA increases observed
post BFR20 exercise exceeded that of FF20 by 50% and the
intracellular volume increases were higher, post exercise R2
changes were similar for BFR20 and FF20. Likewise, after
FF20 exercise, muscle CSA increases and estimated intracel-
lular volume changes were similar to those observed after
FF20WM, yet changes in R2 values were significantly higher.
This could indicate that not all of the BFR induced fluid
shifts contribute to the acute increase in muscle CSA have a
substantial effect on R2. Interestingly, Farup et al. (11) dem-
onstrated that when exercise bouts were repeated over sev-
eral weeks, the elevated acute changes in muscle CSA (i.e.,
swelling) observed with BFR compared to free-flow exercise
conditions were diminished. The primary mechanism lead-
ing to changes in R2 during exercise involves the modulation
of pathways regarding protein-water interactions (57).
Although the osmotic water shifts during exercise are a pri-
mary factor (30, 33, 57), pH, metabolite concentrations, or
other physical factors within the intracellular space that
influence this interaction may contribute as well (32, 57).
Acute increases in CSA also include the increased volume of
the extracellular space which has been demonstrated to have
little impact on muscle R2 values as determined by either
both the mono exponential fit used in this study or from
multi exponential fitting (30, 34, 58, 59). Sjøgaard et al. (60,
61) found that intracellular water volume remained rela-
tively stable during submaximal exercise, while increasing
only during maximal intensity exercise which was accred-
ited to a fluid shift driven by an increasing osmotic gradient
between the extra- and intracellular space. In the same
study, the authors reported acute gains in intracellular water
increases to be both smaller than the concurrent increases in
the extracellular compartment and more varied between
participants, with actual decreases being noted in two of
their six participants. A similar pattern was observed in the
present study, with significant increases in the estimated
extracellular volume across all protocols with smaller
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estimated intracellular volume increases that only reached
statistical significance following BFR20 exercise. The
increases noted were comparable to the 2% increases of mus-
cle water reported after acute low-load (60 W) knee extensor
exercise to exhaustion (62). Even larger increases of intracel-
lular water have been reported during isolated knee extensor
exercise (>10%) but were observed to decrease within
minutes of exercise stop towards values from the earliest
time points measured in the present study obtained 4 min
post exercise (60). Using muscle biopsy sampling, Sjøgaard
et al. (61) estimated the extracellular water fraction to be 12%
for the human quadriceps and also observed that absolute
extracellular water volume increased by 82% following
intense exercise which was concluded to be driven by hydro-
static forces from increased blood flow. These values corre-
spond well with the estimates of the extracellular
compartment in the present study of 10.6% at rest and 16.5%
4 min after exercise cessation. Previous estimates using the
slow R2 compartment to estimate the extracellular fraction
for resting muscle match the present study (10.6%) with val-
ues ranging from 8% to 12% have been reported using vari-
ous models (63–65). The baseline R2 values in this study of
29 s1 are not only similar to reported values from previous
studies which applied monoexponential fits (29 s1 and
33 s1) (53, 63), but are also similar to the primary “intracellu-
lar” component reported from studies using multiexponen-
tial fits (31.2 s–1, Ref. 63, or 31 s–1, Ref. 58). Likewise, the
present changes in R2 after the four different acute exercise
protocols were in the same range as reported earlier for free-
flow muscle exercise (27, 28, 34, 53).
Methodological Considerations
A number of potential methodological limitations should
be taken into consideration for the present study. First of all,
this study only included young males in an attempt to mini-
mize variation between participants at the cost of the study
being less representative of the broader population. Even
then, ensuring the same conditions surrounding each of the
exercise protocols to avoid variations in the physiological
stimulation to participants was challenging. To ensure a uni-
form BFR stimulus we regulated cuff occlusion pressure to
participants based on their individual diastolic blood pres-
sure. Still inter-individual variations were evident, as the
number of repetitions performed until failure with 20% 1RM
in free-flow (FF20) and BFR (BFR20) varied greatly. By using
a cuff pressure of participants' diastolic blood pressure plus
30 mmHg, the resulting mean cuff pressure of 102 mmHg
was slightly below levels of 110 mmHg which have been
recommended since the completion of this study (66).
As previously discussed, the identification of physiological
compartments using R2 values lacks a methodologically pro-
ven model for the present exercise conditions. In the present
study, spin echo times of less than 100 ms were employed to
reduce scan time and increase the number of interleaved
data recordings. This limits the ability to characterize the R2
values of the slower relaxing components which instead
are collected as a constant in a monoexponential fit.
Multiexponential models to include fast R2 components
have been found to provide better fits to T2-weighted signal
on a voxel basis by identifying a fraction of muscle water
 BFR ACCENTUATES POSTEXERCISE mfMRI SHIFTS
(8%–15%) having fast relaxation (R2 > 200 s1), believed to
represent water bound to macromolecules (67). Diffusion
data which include a wider range of b values have also
shown promise separating vascular, extracellular and in-
tracellular compartments (68). A last consideration regard-
ing R2 estimation is the choice of repetition time due to
the R1 relaxation. Sharafi et al. (67) evaluated this effect
using Bloch simulation finding a R2 estimation error
between using a TR of 1,500 ms (as was used in this study)
compared to a TR of 5,000 ms to be 5%. This error was pre-
fered to the considerably longer acquisition time of using
a TR of 5,000 ms.
Future Perspectives
An improved understanding of the physiological stressors
that stimulate muscle hypertrophy plays an important role
in the ability to personalize rehabilitation for patients as well
as training for healthy individuals including top-level ath-
letes (69). Should metabolic stress and associated fluid shifts
drive hypertrophy, or even be significant correlation to it,
magnetic resonance imaging (MRI) would be a highly suited
modality to quantify and map the degree of stimulation in
muscle tissue. The MRI sequences applied in the present
study are clinically accessible and can be interleaved with
other measures to further investigate other physiological pa-
rameters of interest such as oxygen tension (70), perfusion or
specific solute concentrations (71) to study different training
strategies. There are several other desirable adaptations that
skeletal muscles undergo in response to exercise that can be
accentuated by adjusting the training protocol used. For
instance, heavy loads are consistently shown to yield maxi-
mal increases in strength (10, 72) and improve neural recruit-
ment (73) compared to BFR (74). On the other hand, BFR
exercise has been reported to induce a high proliferation of
myogenic stem cells (13, 75, 76), stimulate angiogenic signal-
ing (77), and even induce positive exercise effects in remote
muscle tissues distally to the BFR trained limb (78, 79). The
use of multimodal MRI to examine selected peripheral physi-
ological variables during ongoing exercise represents a
promising tool not only for decoding important mechanisms
for these adaptations but also for optimizing the design of
effective training modalities.
Conclusions
The present MR imaging protocols were able to differenti-
ate the acute exercise-induced response of differing training
protocols to failure that induce skeletal muscle hypertrophy.
The mfMRI measures obtained exhibited larger changes and
longer recovery periods in the exercise protocols designed to
cause elevated metabolic muscle stress. The increase of the
intracellular space was higher following exercise with BFR
compared to free-flow exercise. These findings contribute to
explain the myogenic potential of low-load resistance exer-
cise, with and without BFR, in human skeletal muscle. Thus,
use of multimodal MRI to examine selected peripheral physi-
ological variables during and following acute muscle exer-
cise represents a promising tool not only for decoding
important stressors influencing myocellular adaptation but
also for optimising the design of effective anabolic exercise
modalities.
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DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by
the authors.
AUTHOR CONTRIBUTIONS
B.H., S.K.H., U.L., J.L.N., U.F., P.A., H.B.W.L., and C.S. conceived
and designed research; B.H., S.K.H., U.F., P.A., and C.S. performed
experiments; B.H., S.K.H., and C.S. analyzed data; B.H., S.K.H.,
U.L., J.L.N., P.A., H.B.W.L., and C.S. interpreted results of experi-
ments; B.H. prepared figures; B.H., S.K.H., J.L.N., P.A., and C.S.
drafted manuscript; B.H., S.K.H., U.L., J.L.N., U.F., P.A., H.B.W.L.,
and C.S. edited and revised manuscript; B.H., S.K.H., U.L., J.L.N.,
U.F., P.A., H.B.W.L., and C.S. approved final version of manuscript.
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