BFR Exercise: Muscle Adaptations & Blood Flow Restriction

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Impact of blood flow-restricted bodyweight exercise on skeletal muscle
adaptations
Article in Clinical Physiology and Functional Imaging · February 2018
DOI: 10.1111/cpf.12509
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Clin Physiol Funct Imaging (2018) 38, pp965–975
doi: 10.1111/cpf.12509
Impact of blood flow-restricted bodyweight exercise on
skeletal muscle adaptations
J. E. Jakobsgaard1
1
, M. Christiansen1, P. Sieljacks1, J. Wang1, T. Groennebaek1, F. de Paoli2 and K. Vissing1
Section for Sport Science, Department of Public Health, and 2Department of Biomedicine, Aarhus University, Aarhus, Denmark
Summary
Correspondence
Kristian Vissing, Section for Sport Science, Department of Public Health, Aarhus University, Dalgas
Avenue, 4 DK-8000, Aarhus C, Denmark
E-mail: [email protected]
Accepted for publication
Received 6 December 2017;
accepted 16 January 2018
Key words
angiogenesis; blood flow occlusion; low-intensity
resistance training; muscle metabolism; strength
training
This study ascertains the ability of bodyweight blood flow-restricted (BFR) exercise training to promote skeletal muscle adaptations of significance for muscle
accretion and metabolism. Six healthy young individuals (three males and three
females) performed six weeks of bodyweight BFR training. Each session consisted
of five sets of sit-to-stand BFR exercise to volitional failure with 30-second interset recovery. Prior to, and at least 72 h after training, muscle biopsies were taken
from m. vastus lateralis to assess changes in fibre type-specific cross-sectional area
(CSA), satellite cell (SC) and myonuclei content and capillarization, as well as
mitochondrial protein expression. Furthermore, magnetic resonance imaging was
used to assess changes in whole thigh muscle CSA. Finally, isometric knee extensor muscle strength was evaluated. An increase in knee extensor whole muscle
CSA was observed at middle and distal localizations after training (32% and
35%, respectively) (P<005), and a trend was observed towards an increase in
type I fibre CSA, whereas muscle strength did not increase. Additionally, the number of SCs and myonuclei associated with type I fibres increased by 657% and
20%, respectively (P<005). No significant changes were observed in measures of
muscle capillarization and mitochondrial proteins. In conclusion, six weeks of
bodyweight-based BFR exercise promoted myocellular adaptations related to muscle accretion, but not metabolic properties. Moreover, the study revealed that an
appropriate total training volume needs further investigation before recommending bodyweight BFR to patient populations.
Introduction
Efficient strategies towards counteracting muscle wasting and/
or promoting muscle accretion are potentially desirable in
many clinical settings. Such settings may include individuals
recovering from acute incidents (such as stroke or surgery),
chronic inflammatory diseases (such as rheumatoid arthritis)
and/or increased age (Evans, 2010; Cohen et al., 2015). Resistance exercise has been extensively proven to constitute an
effective mean in promoting muscle myofibrillar protein
accretion and muscle hypertrophy (Folland & Williams,
2007). In this regard, high-intensity resistance exercise (HRE)
(i.e. ≥70% of 1 repetition maximum) is traditionally recommended as the optimal approach (American College of Sports
Medicine, 2009). However, utilization of high mechanical
loads may not be readily applicable in some of the abovementioned clinical settings. It is therefore interesting that lowintensity resistance exercise (LRE), with and without simultaneous blood flow restriction (BFRE), has been shown effective
in promoting muscle hypertrophy and gains in muscle
strength when performed to volitional failure (Scott et al.,
2014; Fahs et al., 2015). Furthermore, BFRE has been proposed as an effective, yet mechanically gentle strategy to promote muscle accretion (Scott et al., 2014), as it requires
substantially less work performed compared to free-flow LRE
(Farup et al., 2015b). This efficiency of BFRE has been suggested to partly rely on an accelerated proliferation of myogenic stem cells (satellite cells, SC) and subsequent addition of
myonuclei (Nielsen et al., 2012). In accordance, maintenance
or expansion of the myocellular SC pool may be of importance for counteracting muscle wasting (Pallafacchina et al.,
2013).
Importantly, improvements of muscle function do not
exclusively rely on accretion of myofibrillar proteins, as
proteins related to muscle metabolic properties also contribute to attainment of such improvements. In accordance,
the impact of HRE on metabolic muscle adaptations has
received relatively less attention (Groennebaek & Vissing,
2017). Yet, recent studies suggest that HRE can stimulate
mitochondrial protein synthesis (Wilkinson et al., 2008) and
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
965
966 Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al.
mitochondrial respiration (Pesta et al., 2011; Porter et al.,
2015). Consequently, it can be assumed that even intermittent alterations in muscle oxygen availability and/or
myocellular homoeostasis with HRE can drive muscle mitochondrial biogenesis. In this regard, Burd et al. (2012)
observed increased mitochondrial protein synthesis after single-bout LRE performed to volitional fatigue, which can be
presumed to accentuate the magnitude of metabolic stress
compared to HRE. In accordance, as BFRE entail both low
intensity and more pronounced ischaemia (Downs et al.,
2014), it can be speculated that BFRE conducted to a state
of fatique possess substantial ability to stimulate mitochondrial biogenesis. This contention is supported by observations that BFRE can improve muscular endurance capacity
(Kacin & Strazar, 2011). Moreover, such adaptations may
be partly reliant on alterations in the capacity of peripheral
blood flow and oxygen delivery to the trained muscle
through increased angiogenesis (Takarada et al., 2002; Evans
et al., 2010; Kacin & Strazar, 2011), with vascular endothelial growth factor (VEGF) proposed to play a central role in
exercise-induced angiogenesis (Hoier & Hellsten, 2014).
Previous BFRE strategies have predominantly involved isolated exercises such as knee extensions, knee flexions and leg
press (Loenneke et al., 2012), which require the use of sophisticated training equipment. However, from a socioeconomic
and safety point of view, it would entail perspective if bodyweight-based BFRE could be successfully practiced at home
during patient rehabilitation.
However, as the effects of bodyweight BFRE have not been
thoroughly investigated, the purpose of this study was therefore to explore the feasibility of this approach in healthy individuals. More specifically, we aimed to investigate the skeletal
muscle adaptational effects of bodyweight BFR training, using
a simple sit-to-stand exercise model. Our hypothesis was that
prolonged bodyweight BFRE is capable of driving adaptations
of importance for muscle metabolic as well as muscle contractile properties.
Materials and methods
Participants
Six healthy young subjects were included in the study [male
n = 3, female n = 3; age 258 years (242; 275); height
174 cm (173; 176); body mass 734 kg (701; 767)] (values
are means and 95% confidence intervals). None of the subjects
had participated in resistance-type training for a minimum of
6 months prior to enrolment in the study. All included female
participants were using oral contraceptives during the intervention period. Each participant was informed of the risks
associated with the study, and a written informed consent was
obtained from each subject prior to participation. The study
was performed in accordance with the Declaration of Helsinki
and approved by The Central Denmark Region Committees on
Health Research Ethics (j. no. 49769).
Study design
In total, the duration of the study was 8 weeks. Prior to, and
following the 6 weeks of training, the subjects underwent a
one-week testing protocol designed to evaluate changes in
exercise capacity and in morphological and myocellular properties. The training period consisted of total 19 sessions with
an average of three weekly sessions dispersed over a six-week
period with at least 24-48 h of recovery between exercise sessions (i.e. progression from two to four sessions per week).
Biopsies were taken from m. vastus lateralis 1 week prior to
the first training session and at least 72 h after the last training
session. Whole muscle CSA was measured by magnetic resonance imaging (MRI). Strength tests consisted of maximal unilateral isometric knee extensor contractions performed on a
dynamometer. Biopsies, MRI and strength tests were performed on three separate days in the stated order both preand postintervention, to prevent interference of the strength
test with morphological and myocellular measures.
Training protocol
Each exercise session began with a 5 minute light-moderate
intensity warm-up on a stationary bike ergometer (Monark
Ergomedic 818E, Monark, Varberg, Sweden). Following the
warm up, a curved 10-cm-wide tourniquet cuff (VBM Medizintechnik, GmbH, Germany) was wrapped around the proximal most portion of the thigh on each subject. The cuffs were
then inflated by a digital tourniquet system with automatic
regulation of pressure (Digital Tourniquet 9000, VBM, Medizintechnik, GmbH, Germany).
Each exercise session was supervised and consisted of five
sets of sit-to-stand BFR exercise with repetitions to voluntary
failure and 30 s of inter-set recovery. The sit-to-stand exercise
was performed by squatting down to a chair and standing up
again. The subjects were instructed to keep their chest high,
back straight and to control the pace of the movement. The
chair was simply used as a marker of depth and safety precaution, making the subjects only lightly touching the chair
before commencing the upward movement of the exercise.
The height of the chair was individually set by placing plates
under the feet of the participants, securing a 90-degree movement of the knee joint for all subjects. A metronome was set
to 60 beats per minute, and the subjects were instructed to
perform each repetition in a four-second duty cycle, making
the downward and upward phases last 2 s each. A set was finished when a subject could not voluntarily initiate upward
movement of another repetition.
During the first training sessions, the cuffs were inflated to
a cuff pressure of 100 mmHg (Nielsen et al., 2012; Farup
et al., 2015b). However, as this cuff pressure entailed a high
number of repetitions, it was seemingly not able to sufficiently reduce arterial occlusion pressure (AOP) in an upright
body position (see Discussion). Therefore, during later training sessions (session 11–19), a higher pressure of 180 mmHg
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al. 967
was applied in the first 2 sets, whereupon it was lowered to
150 mmHg for remaining three sets.
sections (2/3) of the femur length, defined from the top of
caput femoris to the knee joint line.
Determinations of isometric muscle strength
Muscle biopsy
Isometric muscle strength of the knee extensors was evaluated
using an isokinetic dynamometer (Humac Norm, CSMI,
Stoughton, USA). Prior to testing, the same warm-up routine
as mentioned above, was performed. Subjects then seated in
the dynamometer with 90-degree of hip flexion and restraining straps placed around the torso and hips. The axis of rotation of the knee joint was aligned with the axis of rotation of
the lever arm. The lever arm was attached to the subject’s leg
about 3 cm proximal to the medial malleolus. The dominant
leg was used in all strength tests.
With a fully extended knee joint being equal to 0° of flexion, isometric maximal voluntary contraction (MVC) was
measured at 30°and 70° of flexion. The subjects were
instructed on how to avoid countermovement actions and to
contract as fast and forcefully as possible. Subjects were given
four trials, interspaced by at least 1 min of recovery. However, if a subject continued to approve, additional attempts
were allowed. Verbal and visual encouragement was given
during all tests. Torque data were recorded at 200 Hz, and
the highest peak torque of the four attempts was used for further analysis.
Muscle biopsies were obtained before and after the training
period from the middle section of the vastus lateralis muscle
on the dominant leg, using the Bergstr€
om needle technique
(Bergstrom, 1975) and a part of the sample was then treated
for immunohistochemistry, as previously described (Farup
et al., 2015a). The rest of the sample was immediately frozen
in a bath of liquid nitrogen. Samples were then stored at 80°C until further analysis.
Magnetic resonance imaging protocol
Whole muscle CSA was determined from the non-dominant
leg of the subjects using images obtained via a 3 T magnetic
resonance imaging (MRI) scanner (Siemens Skyra, Erlangen,
Germany). The non-dominant leg was chosen to ensure that
no influence from other tests affected the CSA. The subject
was placed in supine position and transported feet first into
the scanner. Two 18-element anterior coils were placed over
the thighs of the subject. A frontal and sagittal localizer scan
was made to localize the knee joint line. Two consecutive
transversal multi-slice fast-spin-echo Dixon scans consisting of
30 slices each with a width of 3 mm distanced 7 mm
between slices were made to ensure CSAs of the full length of
the thighs. Parameters were the following: Field of
view = 450 9 267 mm; acquisition matrix = 384 9 182;
TR = 979 ms; TE = 100 ms; echo-train-length = 5: pixel
bandwidth = 305 Hz/pixel; and number of averages = 1.
The CSA was measured using a custom software program
(Siswin, v09). Three muscle compartments were defined: (i)
the knee extensor compartments (mm. vastus lateralis, vastus
medialis, vastus intermedius and rectus femoris); (ii) the knee
flexor compartment (mm. semitendinosus, semimembranosus,
sartorius and biceps femoris-caput longum and caput breve);
and (iii) the hip adductor compartment (mm. adductor magnus, adductor longus and gracilis). Whole muscle CSA of the
knee extensor muscle compartment was obtained from slices
corresponding to the proximal (1/3), middle (1/2) and distal
Immunohistochemistry
Satellite cell and myonuclei analysis
The preparation of biopsies for immunohistochemestry were
performed by readjusting the samples to 18°C in a cryostat
(Leica CM-3050-S, Leica Biosystems Nussloch GmbH, Nusslock, Germany). Then, serial cross sections of 10 lm thickness
were cut and subsequently placed on microscope glass slides
(Thermo Scientific, Menzel-Gl€azer, Superfrost Ultra Plus).
Muscle biopsy sections were fixed in Histofix (Histolab,
Gothenborg, Sweden) for 4 min and subsequently blocked for
15 h in blocking buffer (2% BSA, 5% FBS, 2% goat serum,
02% Triton x-100, 01% sodium azide).
The sections were then incubated overnight at 4°C in Pax7
primary antibody (1:500; cat. # MO15020, Neuromics,
Edina, MN) followed by incubation in secondary Alexa flour
568 goat anti-mouse antibody (1:200; cat. # A11034, Invitrogen A/S, Taastrup) for 15 h. Afterwards, sections were
incubated in appropriate antibodies for MHC-I (cat. #
A4951, Developmental Studies Hybridoma Bank) and Laminin (cat. # Z0097, Dako Norden) both in 1:500 for 2 h and
secondary antibodies Alexa-Fluor 488 goat anti-mouse green
and Alexa-Fluor 488 goat anti-rabbit green (cat. # A11031
and A11034, Invitrogen A/S, Taastrup, Denmark) both in
1:500 for 1 h. Finally, a mounting media containing 40 ,60 diamidino-2-phenylindole (DAPI) was utilized to visualize
nuclei (Molecular Probes Prolog Gold anti-fade reagent, cat. #
P36935, Invitrogen A/S, Taastrup, Denmark). The stained sections were stored at 20°C until further analysis. All antibodies where diluted in 1% BSA.
SCs were characterized by co-localization of Pax7 and DAPI
within the SC-niche between the basal lamina and the sarcolemma of distinct muscle fibres. Only Pax7/DAPI+ nuclei
with a geometric centre within the basal lamina was considered myonuclei (Fig. 1). SCs and myonuclei were quantified
in a fibre type-specific manner, dependent on association to
type I (A4951+) or type II (A4951) and normalized to
respective number of fibres counted. Finally, myonuclear content was expressed relative to fibre type-specific CSA as a measure of myonuclear domain.
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
968 Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al.
Figure 1 Immunohistochemistry staining. A
representative image of the immunohistochemistry staining on muscle cross sections
for Pax7 (red), MHC-I (green) and DAPI
(blue). Lower right corner displays a magnified panel with the arrows specifying two
identified Pax7+ nuclei (SCs) associated with
type I fibres. [Colour figure can be viewed at
wileyonlinelibrary.com]
All images were obtained at 209 magnification with fluorescents microscope with a Leica DFC450 camera attachment
utilizing Leica Application Suite and subsequently analysed
using ImageJ software (v. 149). To ensure reliable numbers
of SCs and myonuclei, a mean of 145 [130; 160] and 93
[86; 100] fibres for each subject at each time point were
assessed, respectively (mean and 95% confidence interval).
area and capillarization quantification, a mean number of 395
fibres [382; 408] for each subject at each time point was
analysed. The same blinded investigator conducted the quantification of SC’s and myonuclei content as well as CSA and
capillaries to ensure a uniform procedure. Fibres situated on
the edge of the cross sections as well as fibres characterized
by poor morphological integrity where excluded from quantification.
Fibre area and capillarization
With regard to fibre CSA and capillary analysis, biopsies were
prepared as initially described above. After the initial blocking,
the sections were incubated overnight at 4°C in primary antibody for MHC-I (1:1000; cat. # A4951, Developmental Studies Hybridoma Bank). Subsequently, sections were incubated
with Alexa-fluor 568 goat anti-mouse red (1:500; Molecular
Probes, cat. no A11001, Invitrogen A/S, Taastrup, Denmark)
and Alexa-fluor 488 mouse-anti-Human Collagen IV green
secondary antibodies (1:100; cat. # 53-9871, Affymetrix, CA,
US). Finally, a cover slip was applied onto the glass slides
using a mounting medium not containing DAPI (Molecular
Probes Gold anti-fade reagent, cat # P36930, Invitrogen A/S).
Samples were stored at 20°C until further analysis. All antibodies where diluted in 1% BSA.
Images were captured at 910 magnification with a Leica
fluorescent microscope, and analysis was carried out using
Leica QWin software (v. 351). Fibre CSA for two major fibre
types were assessed and expressed as mean values relative to
both type I and type II fibres, separately. The capillaries
around each fibre were assessed both relative to total number
of fibres counted and in a fibre type-specific manner. Furthermore, the total number of capillaries was expressed relative to
fibre type CSA as a measure of capillary density. For fibre type
Immunoblotting
Preparation of muscle lysates and determination of protein
concentration was conducted as previously described (Rahbek
et al., 2015). Equal amounts of protein were separated by SDSPAGE on precast Stain-Free 4%–15% gels (Bio-Rad, CA, USA)
running for approximately 45 min at 160 V. Proteins were
then electro-blotted onto PVDF membranes (Bio-Rad, CA,
USA) using a Trans-Blot Turbo system (Bio-Rad, CA, USA).
Control for equal loading was performed using the Stain-Free
technology as previously described (Gilda & Gomes, 2013;
Gurtler et al., 2013). Membranes were then blocked for 2 h in
01% I-block (Applied Biosystems, CA, USA) diluted in TBST
and incubated overnight with the respective primary antibodies. The following primary antibody was purchased from Cell
Signaling Technology (Danvers, MA, USA) and utilized as follows: COXIV (cat. # 4844, rabbit polyclonal, conc. 1:1000 in
5% skim milk). The following primary antibodies were purchased from ABCAM (Cambridge, UK) and utilized as follows:
CPT1B (cat. # AB134135, rabbit monoclonal, conc. 1:1000 in
5% BSA), Citrate Synthase (cat. # AB96600, rabbit polyclonal,
conc. 1:1000 in 5% BSA), VEGF-A (cat. # AB46154, rabbit
polyclonal, conc. 1:500 in 5% BSA), b-HAD (cat. # AB81492,
rabbit polyclonal, conc. 1:1000 in 5% BSA). With regard to
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al. 969
secondary antibodies, membranes were then incubated 1 h
with Horseradish peroxidase-conjugated goat anti-rabbit (cat.
# 2054, Santa Cruz, TX, USA) conc. 1:5000 in 1% BSA. All
antibodies were diluted in TBST. Proteins were visualized by
chemiluminiscence (Thermo Scientific, MA, USA) and quantified with UVP VisionWorks LS software (UVP, CA, USA). Precision Plus Protein All Blue standards were used as markers of
molecular weight (Bio-Rad, CA, USA). Arbitrary intensity data
of each target were normalized to total protein obtained from
the Stain-Free blotting technology (Gurtler et al., 2013).
Statistical analysis
Differences from pre- to post-intervention in repetitions, muscle strength, knee extensor whole muscle CSA, protein expression, capillary, myonuclei and SC were determined using a
paired t-test. With regard to analysis of protein expression,
arbitrary intensity data of each target were normalized to total
protein obtained from the Stain-Free blotting technology
(Gurtler et al., 2013) before being log-transformed to reach
normal distribution prior to analysis. Statistical analysis was
performed using SigmaPlot (v120, Systat Software, San Jose,
CA). Alpha level was set to P≤005.
Results
Training progression
All six subjects completed all training sessions. Fig. 2 shows
total repetitions during the first and last training session in
each period (i.e. session 1–10 and sessions 11–19 with
100 mmHg and 180 mmHg of applied cuff pressure, respectively). An increase of 119 [41; 197] and 80 [6; 155] in the
total number of performed repetitions was observed from the
first to the last training session for the 100 mmHg and
180 mmHg periods, respectively (P<005).
Muscle growth and strength
An increase in knee extensor whole muscle CSA was observed
after training, at both the middle (+24 cm2 [13 cm2;
35 cm2]) and distal (+25 cm2 [11 cm2; 38 cm2]) sections,
corresponding to increases of 32% and 35%, respectively
(P<001). No changes were observed at the proximal section
(Fig. 3a). Also, no changes in whole muscle CSA of the
adductor and flexor muscle groups were observed at any of
the measured localizations (not presented). With regard to
muscle fibre area, no changes were observed from pre- to
post-intervention of either type I or type II muscle fibres
(Fig. 3b). No changes in isometric knee extensor strength
were observed at either joint angle (Fig. 3c).
SCs and myonuclei
We observed an increase of 004 [000; 008] in SCs per type
I fibre from pre- to postintervention, corresponding to an
increase of 657% (P<005) (Fig. 4a). No changes in SC per
type II fibre were observed.
With regard to myonuclei, an increase of 032 [001; 063]
in mean number of myonuclei associated with type I muscle
fibres was observed from pre to post, corresponding to an
increase of 20% (P<005). No changes for myonuclei associated with type II fibres were observed (Fig. 4c). A decrease of
361 lm [699 lm; 23 lm] (107%) in myonuclear
domain for type I fibres was also observed (P<005), with no
changes for type II fibre myonuclear domain (Fig. 4d).
Angiogenesis
No changes were observed in capillary density or in number
of capillaries per fibre ratio (Fig. 5).
Metabolic proteins and VEGF-A
Western blots are shown in Fig. 6a. No changes were
observed with training in protein content for any of the protein targets (Fig. 6b).
Discussion
Figure 2 Total repetitions as training progression. Total repetitions
performed in the first and last training session in each period
(100 mmHg and 180 mmHg, respectively). Data are presented as
mean SEM. *Denotes a difference between the first and last session
with same pressure (P<005).
The purpose of the current study was to investigate a potential
dual effect of 6 weeks of bodyweight-conducted BFRE on
adaptations of significance for skeletal muscle contractile and
metabolic properties. The study population comprised healthy
young individuals as a model to provide a preliminary investigatory platform for future studies on how to counteract decay
in muscle health in populations experiencing skeletal muscle
loss.
The main finding of the current study was that our
approach was capable of promoting phenotype-specific adaptations related to muscle growth, with type I fibres exhibiting
the more pronounced adaptations. Contrary, little effect was
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
970 Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al.
Figure 3 Muscle growth and maximal isometric strength. Mean SEM (bars) and individual changes (lines) in knee extensor whole muscle CSA
as measured by MRI (a), and mean SEM and individual changes of fibre type-specific area as determined by immunohistochemistry (b). Maximal isometric knee extensor muscle strength measured at 30° and 70° knee flexion pre- and postsix weeks of bodyweight sit-to-stand exercise
under BFR (c). Values are presented as mean SEM (bars) and individual changes (lines). **Denotes a change from pre to post (P<.01).
observed on adaptations related to skeletal muscle metabolism.
Additionally, our study simultaneously advances some questions on how a bodyweight BFRE protocol should be designed
to minimize the work volume required to produce such muscle adaptations.
Impact of bodyweight BFRE on muscle protein accretion
and strength
The training protocol provoked a significant increase in whole
muscle knee extensor CSA measured at the middle and distal
locations (32% and 35%, respectively), with no changes
observed at the proximal portion. Such increases in whole
muscle CSA of the knee extensors are within range of what
has previously been observed after single-joint BFRE exercises
in studies using comparable magnitude of training (Wernbom
et al., 2008). A lack of, or impaired growth in whole muscle
knee extensor CSA at the proximal portion of the femur, has
previously been observed by Kacin & Strazar (2011) and Ellefsen et al. (2015) after knee extensor BFRE. This prompts speculations on whether the external pressure of the cuff itself
may somehow negate localized muscle growth in the proximity of the cuff. Furthermore, the lack of muscle hypertrophy
in knee flexor and hip adductor compartments indicates that
the work load was primarily imposed on the knee extensor
compartment, which immediately question if the occlusion
was equally restricting blood flow to the different muscle
compartments and/or if the BFRE sit-to-stand exercise alone is
sufficient to affect thigh flexor and adductor compartments.
On muscle fibre level, no significant changes in CSA were
observed, although a non-significant ~85% mean increase in
type I fibre area was observed, with four of the subjects displaying even more pronounced increases in type I fibre CSA.
The observed magnitude of discrepancy between changes in
whole muscle CSA and fibre CSA is similar to previous observations (Aagaard et al., 2001; Farup et al., 2012), thus supporting that the observed increase type I fibre CSA is indeed valid.
The increases in CSA likely resemble true increases in muscle
myofibrillar protein accretion, as water retention has previously been observed to return to baseline within few hours
after BFRE (Farup et al., 2015b). Given the volume of lowintensity work in the present model, the muscle fibre
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al. 971
Figure 4 Satellite cells (SC) and myonuclei. Mean number of SC per fibre type is presented as mean SEM (a) and individual data (b). Mean
number of myonuclei per fibre type is presented as mean SEM (bars) and individual changes (lines) (c). Measures of myonuclear domain (fibre
area per myonuclei) are presented as mean SEM and individual changes (d). *Denotes a change from pre to post (P<005).
Figure 5 Angiogenesis. Measures of capillary density represented as mean SEM (bars) and individual changes (lines). Mean number of capillaries per fibre ratio is presented as mean SEM and individual changes (b).
recruitment may have relied greatly on type I fibres, providing
a feasible explanation for a hypertrophic response in this fibre
type. Furthermore, the finding of a clear increase in the number of myonuclei supports that type I fibre hypertrophy did
indeed occur. Accordingly, one possible explanation for
increased myonuclear number relates to a myocellular
necessity to increase transcriptional capacity to maintain a
finite protein turnover of a given fibre size (Petrella et al.,
2008). Alternatively, increased myonuclear numbers may
relate to regenerative processes in response to exerciseinduced muscle micro-damage (Paulsen et al., 2012). In accordance, the fact that we also observe an increase in SCs in type
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
972 Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al.
Figure 6 Immunoblotting results. Blots of all protein targets for all subjects (n = 6) from muscle biopsies obtained before and after training (a).
All blots for one target was performed on the same gel. Changes in protein expression of muscle metabolic proteins and VEGF-A are presented as
individual and mean fold changes SEM from pre- to post-training (b). CS, citrate synthase; b-HAD, 3-hydroxyacyl-CoA dehydrogenase; COXIV,
cytochrome C oxidase subunit IV; CPT1B, carnitine palmitoyltransferase 1B; VEGF-A, vascular endothelial growth factor A.
I fibres could reflect ongoing regenerative processes due to
the volume of work these fibres were exposed to. Interestingly, different subpools of SCs are believed to have different
faiths (Yin et al., 2013), and it has been proposed that
increases in myonuclear abundance through SC proliferation
can precede substantial myofibre growth, resulting in a temporary decrease of the myonuclear domain (Gundersen,
2016), similar to what we observe in the current study. In
this regard, although a previous study from our laboratory
demonstrated that unaccustomed BFRE performed to volitional
failure could produce muscle-damaging responses, these
responses were attenuated upon repeated exposure single-bout
BFRE, indicating that muscle damage is negligible in the
trained state (Sieljacks et al., 2016).
It may seem immediately puzzling that muscle growth was
not accompanied by improvement in muscle strength. However, a lack of changes in isometric muscle strength despite
muscle growth has previously been demonstrated after lowload BFR training (Kacin & Strazar, 2011), indicating that muscle growth and gains in muscular force capacity as a response
to BFRE may not be closely related. Alternatively, the absence
of muscle strength development in the early days after completion of BFR training has been suggested to relate to a delay in
muscle strength development due to ongoing regenerative
processes after BFR training (Nielsen et al., 2017). In accordance, Nielsen et al. observed no changes in slow velocity isokinetic knee extensor MVC 5 days after training, yet an increase
to emerge 12 days after training. In this regard, it is important
to notice that the training frequency of the Nielsen study comprised 23 sessions dispersed over as little as 19 days of training,
which is very different to the three weekly sessions employed
in our current study. Consequently, the ability and necessity of
the muscle to recover/regenerate seem less influenced in the
current study. On the other hand, it cannot be excluded that a
different timing of strength testing in the current study would
have improved our ability to identify a change. It therefore
seems premature to adjudicate bodyweight-based BFRE as an
insufficient stimulus to improve strength, especially in individuals with low baseline in muscle strength capacity. In this
regard, it is noteworthy that bodyweight-conducted BFRE has
been shown capable to promote increased isometric strength in
elderly individuals (Yokokawa et al., 2008).
Impact of bodyweight BFRE on muscle metabolic-related
properties
Skeletal muscle angiogenesis, as typically demonstrated by
increases in capillary density and number of capillaries per
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al. 973
muscle fibre, is important to facilitate muscle oxygen and
substrate availability and muscle metabolism inherent of aerobe endurance-type exercise (Hoier & Hellsten, 2014).
Interestingly, resistance exercise has also been observed to
stimulate angiogenesis, with the number of capillaries
increasing in proportion to the hypertrophic response, while
the capillary density is unaltered, thus maintaining diffusion
capacity (McCall et al., 1996; Hoier & Hellsten, 2014; Holloway et al., 2017). Contraction-induced ischaemia inherent
of resistance training may adhere to reduced tissue oxygenation (Downs et al., 2014), thus potentially imposing stimulation of angiogenesis through hypoxia-induced signalling
pathways (Hoier & Hellsten, 2014). As ischaemia and
reduced tissue oxygenation can be assumed to be enhanced
with BFRE compared to traditional resistance exercise
(Downs et al., 2014), BFRE can be assumed to promote
angiogenesis. Yet, in the present study, we observed no
changes in capillarization and VEGF-A protein content, but
rather a trend towards a decrease in capillary density among
type I fibres (P = 0064). Furthermore, we observed no
changes in the expression of proteins involved in muscle
mitochondrial metabolism. These results are immediately
surprising, not least because our subjects displayed marked
improvements in endurance capacity, as shown by the gradual increase in total work performed during the training
period. To our knowledge, no previous study has examined
the impact of BFRE on mitochondrial protein content or
mitochondrial respiratory capacity. On the other hand, HRE
has previously been reported to produce, concomitant
increases in muscle fibre area and enzyme activity of mitochondrial enzymes (Tang et al., 2006). Furthermore, in a
recent study by Porter et al. (2015), improvements in mitochondrial function, as measured by high-resolution
respirometry (HRR), were observed after 12 weeks of HRE.
Thus, the capability of BFRE to enhance muscle mitochondrial biogenesis deserves further investigation.
Considerations on usefulness of bodyweight BFRE to
counteract decay in skeletal muscle health in patient
populations
The motivation of the current study was to identify an effective, gentle (i.e., low load) and practical way to stimulate
adaptations related to skeletal muscle growth and metabolism
for populations experiencing loss of muscle mass. In accordance, BFRE strategies may entail a socioeconomic benefit,
provided it can be self-conducted at home without sophisticated training apparatus or supervision. We considered that
the feasibility of such an approach needed to first be
addressed in young healthy individuals. Moreover, we were
curious to see whether our strategy was indeed capable of
inducing robust adaptations even in a rather small and
heterogenic population, as this is often most expectedly a
challenge to experience when conducting human studies on
patient populations. By this deliberate approach, we made
some interesting observations. In accordance, all the subjects
completed all training sessions without experiencing adverse
events. The study also serves to emphasize that efforts to
control the training volume necessitate careful considerations
on appropriate cuff pressure, to obtain an appropriate relative degree of occlusion and thereby reduce time to exhaustion. In accordance, the current, as well as most previous,
studies on BFRE have employed absolute fixed cuff pressures
close to 100 mmHg, while some other studies have used
above even 200 mmHg (Scott et al., 2014). In consideration
on potential discomfort associated with high cuff pressures
in future studies on patient populations, we initiated the
training with a cuff pressure 100 mmHg. However, due to
the rapid progress in work volume, we later questioned
whether cuff pressure was sufficiently high to uphold a sufficient relative degree of blood flow occlusion during an exercise such as the sit-to-stand exercise (i.e. multi-joint exercise,
involving several muscles of the lower extremity). In accordance, it has been proposed that absolute cuff pressure
should better consider the effects of gravity on regional
blood pressure in upright compared to supine body positions
(Sieljacks et al., 2017).
Given small sample size and heterogeneity of the
included subjects, the results should be interpreted with
some caution. Oppositely, the stimulatory regime is robust
enough to produce improvements in a setting that mimics
the expected heterogenetic composition of individuals of a
clinical population. In accordance, our findings of statistically verifiable increases in whole muscle CSA and myonuclei with training exhibited by similar directions of change
in all subjects. In previous studies of our laboratory, we
demonstrate that we are indeed experienced and capable in
handling the implemented methods and analytic procedures
(Farup et al., 2014a,b; Rahbek et al., 2014; Farup et al.,
2015a; Larsen et al., 2014).
In principle, it could be criticized that the current study did
not implement a non-BFRE control group. However, whereas
this should be implemented in future studies on a clinical
populations, in the current study on healthy young individuals, the work volume would have been inappropriately high.
In summary, bodyweight BFRE seems capable of promoting
adaptations of significance for muscle contractile properties,
whereas further investigation is needed on its potential for
promoting adaptations of significance for metabolic properties.
Acknowledgments
Julie Moeslund and Freja Holm are thanked for their assistance
with MRI analysis.
Conflict of interest
The authors have no conflicts of interest.
© 2018 Scandinavian Society of Clinical Physiology and Nuclear Medicine. Published by John Wiley & Sons Ltd 38, 6, 965–975
974 Bodyweight blood flow-restricted exercise, J. E. Jakobsgaard et al.
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