Juntendo Medical Journal

Objective: The purpose of this study was to investigate whether blood flow restriction (BFR) walking results in an acute increase in muscle size and decrease in maximal strength as well as metabolic accumulation in older adults. Methods: Ten older men and 8 older women (mean±standard error; age, 68±1 years; height, 161.8±1.8 cm; weight, 60.7±8.5 kg) walked for 20 minutes with BFR at a pre-determined speed (3-4 km/h). Muscle thickness (MT) was measured at the following four sites: knee extensors (KE) and flexors (KF) at 50% of the thigh length and dorsi flexors (DF) and plantar flexors (PF) at 30% of the lower leg length. The maximal voluntary isometric strength of KE and blood lactate concentrations were measured. All parameters were measured before and immediately after the exercise session. Results: MT increased at all sites after BFR walking (KE: pre 28.9 ± 1.0 mm, post 32.3 ± 0.9 mm; KF: pre 53.3±1.2 mm, post 55.2±1.4 mm; DF: pre 23.9±0.6 mm, post 24.7±0.5 mm; PF: pre 61.0±1.1 mm, post 63.8±1.0 mm). The maximal voluntary isometric strength of KE decreased after BFR walking (pre, 144±9.5 Nm; post, 136.5±8.7 Nm). Blood lactate concentration was significantly elevated after BFR walking (pre, 1.3± 0.1 mol/l; post, 1.9±0.2 mol/l). Conclusion: BFR walking causes an acute increase in muscle size and decrease in maximal strength as well as metabolic accumulation in older adults.


Introduction
Rosenberg first proposed the term "sarcopenia" to describe the decrease in skeletal muscle mass with advancing age 1) . Advanced skeletal muscle loss leads to obesity and osteoporosis, as well as a decrease in quality of life and physical performance in daily living 2) . Therefore, maintaining a healthy amount of skeletal muscle mass is important to prevent disability. Generally, high-intensity resistance training is recommended for preventing sarcopenia 3) . The guidelines of the American College of Sports Medicine recommend lifting weight equivalent to at least 70% of one repetition maximum to maximize muscular hypertrophy in older adults 4) .
Recently, blood flow restriction (BFR) has received attention for inducing muscle hypertrophy. Its unique characteristic is that substantial muscle hypertrophy can occur with an intensity as low as 10% of maximal voluntary contraction 5) . Previous studies have found that muscle hypertrophy occurs following walking (about 10% of maximal voluntary contraction 5) ) with BFR in young 6) and older adults 7) . Furthermore, compared to 6 months of non-BFR walking exercise 8) , BFR walking can induce muscle hypertrophy in a very short period (10 weeks) 7) . Therefore, BFR walking may be beneficial to a wider range of the population, promoting muscle hypertrophy at low intensity and in a short time period 9) .
The mechanism of the effect of BFR walking on muscle hypertrophy in exercised muscle is not completely understood. However, a previous study by Ozaki et al. indicated that the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and the dephosphorylation of eukaryotic translation elongation factor 2 (eEF2) after BFR walking in young adults 10) results in the augmentation of the net protein balance 11) . Muscle cell swelling and fatigue (i.e. decreasing muscle strength) caused by accumulation of metabolites 11)-13) are thought to be associated with muscle hypertrophy as upstream stimulatory factors of ERK1/2 and eEF2. A previous study by Ogawa et al. reported that the increase in thigh muscle thickness, an index of muscle cell swelling, was more pronounced with rather than without BFR after walking in young adults 14) . Furthermore, the relative load during walking was decided by the ratio of body weight to leg muscle strength. Muscle strength per body weight is lower in older adults than in young adults 15) . However, the effects of BFR walking on muscle cell swelling and muscle strength in older adults have not been studied so far. We speculate that it induces more muscle cell swelling and muscle strength reduction in older adults than in young adults. Thus, the purpose of this study was to investigate whether BFR walking can elicit acute increase in muscle thickness and decrease in maximal strength as well as metabolic accumulation in older adults.

Subjects
Ten older men and 8 older woman (mean ± standard error; age, 68 ± 1 years; height, 161.8 ± 1.8 cm; weight, 60.7 ± 8.5 kg) participated in this study. The subjects were recruited through printed advertisements. All subjects were informed of the methods, procedures, and risks, and signed an informed consent form before participating in the study. The study was conducted according to the Declaration of Helsinki and was approved by the Ethics Committee for Human Experiments of Juntendo University, Japan.

BFR walking
The walking exercise consisted of 20 minutes of walking at a pre-determined speed (3-4 km/h) on a motor-driven treadmill. A 105-mm-wide nylon cuff (MT-870 Digital Tourniquet, Mizuho, Tokyo, Japan) was applied tightly at the most proximal portion of both the legs. The pressure was calculated for each subject based on the circumference of the right thigh (33% of the distance from the inguinal crease to the top of the patella), as follows: < 50 cm = 100 mmH g (men, n = 9; women, n = 5); 51-55 cm = 120 mmHg (men, n = 1; women, n = 2); 56-59 cm = 40 mmHg (women, n = 1). This is because arterial occlusion pressure is largely influenced by thigh circumference 16) . The cuff air pressure was released immediately upon completion of each session.

Muscle thickness
Before and immediately after an exercise session, muscle thickness was measured via B-mode ultrasound using a 5-18 MHz scanning head (Noblus; Aloka, Tokyo, Japan) at the following four sites as an indirect index of muscle cell swelling: the central surface of the knee extensors (KE) and flexors (KF) at 50% of the right thigh length between the lateral condyle of the femur and the greater trochanter, and the central surface of the dorsi flexors (DF) and the medial surface of the plantar flexors (PF) at 30% of the right lower leg length between the lateral malleolus of the fibula and the lateral condyle of the tibia. Prior to all scans, the subjects rested quietly in a seated position for at least 30 min to avoid an influence of fluid shifts within the muscle,. Circumferences were also measured at 50% of the thigh length and 30% of the lower leg length using a tape measure. The same investigator performed all measurements to maximize intrarater reliability. Ultrasound measurements of muscle thickness were performed in the supine/prone position, with careful attention to ensure that the hip and ankle joint positions and the distance between both legs were the same in all the measurements. The scanning head was coated with a water-soluble transmission gel and placed on each marked measurement site without depressing the dermal surface. The subcutaneous adipose tissuemuscle interface and the muscle-bone interface were identified on the ultrasound images, and the distance between the two interfaces was recorded as the muscle thickness.

Maximal isometric strength
Maximal isometric strength of knee extensors was measured using a Biodex system 4 dynamometer (Biodex Medical Systems, Shirley, NY) before and immediately after an exercise session to determine the degree of muscle fatigue after BFR walking. During testing, each participant was seated on a chair with the hip joint angle positioned at 85°of flexion (0°= full hip extension). The center of rotation of the knee joint was visually aligned with the axis of the dynamometer lever arm and the ankle was firmly strapped to the distal pad of the lever arm. A knee joint angle of 0°corresponded to full knee extension. Several warm-up contractions (4-5 submaximal contractions and 1-2 near-maximal contractions) were performed before testing. Participants were then instructed to perform maximal isometric knee extension for about 5 s at a fixed knee joint angle of 75°. Two maximal efforts were performed for each isometric measurement peak torque and the highest value was used in data analysis.

Blood lactate concentration
Prior to testing, subjects rested quietly in a seated position for at least 30 min. Whole blood samples (0.3 µl) were also taken from the fingertip before and immediately after the exercise session. Blood lactate concentration was determined using lactate oxidase enzyme electrode methods in a lactate analyzer (Lactate pro 2, ARKRAY, Kyoto, Japan). The detection limit was 0.5 mmol/l.

Statistical analysis
Results are expressed as mean ± SE for all variables. First, we performed a two-way analysis of variance (ANOVA) with repeated measures [sex (male and female)× time (pre and post)] to declare sex difference among variables. However, there is no significant interaction within all variables. Therefore, two-tailed paired Studentʼs t-test (pre and post) was collectivity performed the value of older men and older woman as one group. The level of significance was set to p < 0.05.
Changes in the maximal isometric strength of the KE are shown in Figure-3. Maximal isometric strength of knee extensors significantly decreased after BFR walking (pre, 144 ± 9.5 Nm; post, 136.5 ± 8.7 Nm).

Discussion
The main finding of the present study was that walking with BFR elicited acute increase in muscle thickness and decrease in maximal isometric strength as well as a concomitant increase in blood lactate concentration.
In the present study, blood lactate concentration significantly increased by 1.9mmol/l, immediately after BFR walking. Loenekene et al reported that blood lactate concentration increased by 2.0mmol/l following BFR walking in healthy young adults 17) . There has been previous study that investigating blood lactate concentration after 20 minutes of BFR walking at 4.5km/h in older people, which significantly increased from 1.1mmol/l to 1.4mmol/l (Ozaki et al. 2015 in press). These results show that blood lactate concentration increase after BFR walking regardless age. However, compared to other exercise modality such as low intensity resistance training with BFR, the fold change in blood lactate concentration is approximately less than half 18) . The small changes in blood lactate concentration of BFR walking in present study may be attributable to the exercise intensity in addition to differences among exercise modality.
Our results showed that muscle thickness, an index of muscle cell swelling, significantly changed in all evaluated regions following BFR walking in old adults. The percent changes in muscle thickness of KE were approximately 11.7%, which is almost equal to previous study (8.8% and 10.1%, respectively) that investigated the acute increase in muscle thickness following BFR walking in young adults 14) . The precise mechanism for muscle hypertrophy following BFR exercise cannot be ascertained, and it has been speculated that muscle cell swelling plays an important role in gaining training effects. A recent study indicated that acute increase in muscle thickness is correlated with a loss of plasma volume 19) , suggesting that the increase in   muscle thickness after BFR exercise reflects primarily a fluid shift from vascular space into exercised muscle. This may occur as a consequence of increase in metabolites such as Pi + and H + and decrease in pH during muscle contraction 20) 21) . Indeed, direct relationships between accumulation of Pi + and decrease in pH during exercise and muscle hypertrophy following a period of low-intensity resistance exercise with BFR have been reported 22) . Currently, it is theorized that exerciseinduced metabolites mediates muscle protein synthesis via muscle cell swelling, leading to muscle protein signaling, such as through mammalian target of rapamycin and mitogen-activated protein kinase signaling pathway 23) .
Our results showed that maximal isometric strength in older adults decreased significantly by 5.2%, following BFR walking, whereas previous results have shown that maximal isometric strength did not decrease (2.1%) following a bout of walking combined with BFR in young adults 14) . The relative decrease in the lean body mass and muscle strength compared to body mass in older adults may contribute to the acute decline of muscle strength following BFR walking. Janssen et al. reported that age was negatively correlated (p < 0.01) with relative skeletal muscle mass (body mass/skeletal muscle mass) in men (r =-0.50) and women (r = -0.24) 24) . Furthermore, other study reports show that relative maximum muscle strength (muscle strength/body weight) of KE gradually decreases with age 25) . This decline would put a load on exercised muscle during BFR walking in older individuals than in young adults. Furthermore, metabolic accumulation was elevated after BFR walking in our study. Several studies have reported that the accumulation of metabolites such as lactate, Pi, H + , and pH causes a decrease in muscle strength 26)-29) . For example, the rise in whole blood lactate after repeated knee extension and flexion was strongly correlated to the fall in maximal isokinetic strength during exercise (r = 0.822) 29) . Therefore, it is presumed that the accumulated metabolites partly participate in decreasing the muscle strength following BFR walking in older adults.
The limitation of the present study was that there was no control group without BFR. A previous study reported an acute change in muscle thickness in blood-flow restricted quadriceps, but not in non-restricted quadriceps, during and immediately after walking. Thus, we speculate that muscle thickness is not increased after walking in older adults 14) . Furthermore, even though the magnitude of the decrease in isometric strength after BFR walking is associated with cuff compression pressure 18) , the nylon cuff-inflating device did not apply an initial compressive force. Additional research is needed to address these issues.
In conclusion, our results indicated that an acute increase in muscle thickness and a decrease in muscle strength occur following BFR walking as does metabolic accumulation in older adults.