Effect of Combined Increased Physical Activity and Walking with Blood Flow Restriction on Leg Muscle Thickness in Older Adults

Objective : To investigate the effect of combined increased walking and stair-climbing, and walking with blood flow restriction (BFR) in working muscles on leg muscle thickness in older adults. Methods : Nineteen older subjects were divided into the following three groups: normal walking and stair-climbing (WS), WS and BFR-walk once a wk (WS-BFR1), and WS and BFR-walk twice a wk (WS-BFR2). All subjects were instructed to walk at a self-selected, faster pace than usual for ≥ 30 min per session, ≥ 2-4 days per wk, and climb ≥ 5 flights of stairs per day, ≥ 4 days per wk for 11 wk. Additionally, the WS-BFR1 and WS-BFR2 groups performed 20 min of a BFR-walk at a pre-determined exercise intensity of 70-85% of the age-predicted maximum heart rate. Results : Two-way repeated measures analysis of variance showed that the time effects were significant (p< 0.01) for muscle thicknesses (MT) of the posterior aspects of the thigh (PT, WS: 8.9%, WS-BFR1: 11.7%, WS-BFR2: 11.8%) and lower leg (PL, WS: 3.0%, WS-BFR1: 1.1%, WS-BFR2: 6.6%). However, there was no significant difference in these values of MT among all the groups. For the 10-m walking time, the main effect of time was significant (p<0.05), but the interaction was not significant. Conclusions : Walking and stair-climbing training can increase MT of the PT and PL, and improve walking performance in older adults, whereas a BFR-walk once or twice a wk may not produce additional training effects.


Introduction
Skeletal muscle is important for performing activities of daily living, and it has an important role with metabolism. For instance, skeletal muscle is the largest disposal site for ingested glucose 1) , and it plays a role in lipid oxidation and immune responses 2) 3) . The amount of skeletal muscle also decreases with aging, i.e., sarcopenia. This causes disability, falls, and osteoporosis 4)- 6) , and it increases the risk of developing a wide range of chronic disorders, including atherosclerosis 7) 8) , insulin resistance, and hyperglycemia 9) 10) . One of the biggest problems is a reduction of ambulatory ability resulting from the decrease in lower limb muscles. Therefore, strategies to increase or maintain the amount of skeletal muscle, especially in the lower limb, across oneʼs lifespan are important for overall health. High-load resistance training has been primarily recommended as an effective countermeasure against sarcopenia 11) 12) ; in fact, the decline in muscle mass and strength among older adults is at least partly reversible by highload resistance training 13) 14) . However, high-load resistance training generally requires a supervised program involving expensive training equipment, and high external loads may present a major barrier for older adults.
Recently, Abe et al. 15) showed that activities of daily living at moderate and vigorous intensities (i. e., ≥3 Mets) are positively correlated with the triceps surae and tibialis anterior muscle thickness. Additionally, isometric knee flexion strength is positively correlated with the duration of moderate physical activity. Another study reported that 6-month walking training, one of the typical examples of moderate physical activity, increased muscle thickness (MT) in the knee flexors and dorsi flexors, and strength in the knee flexors, dorsi flexors, and plantar flexors; however, there was no significant effect on the knee extensors 16) . Given that age-related changes in MT were a site-specific manner, upper-leg anterior MT especially decreases with age; thus, it is important for older adults to perform other exercise programs to improve muscle size and function in knee extensors as well as walking during daily physical actions. A common example of daily physical action that activates the knee extensors more than walking is stair-climbing 17) . To the best of our knowledge, no study has investigated the hypertrophic effect of stair-climbing.
Physical activities such as walking at moderate to vigorous intensities can induce significant muscle hypertrophy, but the effect is minimal 18) . Muscle hypertrophic adaptations mainly result from exposure to both mechanical and metabolic stresses 19) . Metabolic stress is accumulated by blood flow restriction (BFR) in working muscles even during walking, resulting in an enhanced hypertrophic effect with walking 19) . For example, our studies showed that 20 min of a BFR walk, 4-5 days per wk for 6-10 wk, increased thigh muscle size and strength, whereas these adaptations were not observed after a normal walking program in older adults 20) 21) . It would be difficult, however, for most older adults to perform a BFR-walk at a high frequency as in previous studies 20) 21) because a supervised program with a BFR-dedicated device is required.
To develop a more practical training program, we investigated the effect of combined walking and stair-climbing on almost a daily basis in older adults, and whether replacing 1-2 days per wk with a BFR-walk has an additional training effect on leg muscle size in older adults.

Participants
Twenty-six older subjects (age 69 ± 1 years, height 1.63 ± 0.02 m, body weight 64.5 ± 2.0 kg) volunteered to participate in this study. They were recruited through printed advertisements and by word of mouth. None of the subjects had participated in any regular high-load resistance training for at least 1 year. All subjects were informed about the methods, procedures, and risks, and they provided informed consent before participating in this study. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee for Human Experiments of Juntendo University, Japan. Participants were randomly assigned to the following three groups: normal walking and stair-climbing (WS, n = 16, 12 men and 4 women), WS and a BFR-walk once a wk (WS-BFR1, n = 12, 7 men and 5 women), and WS and a BFR-walk twice a wk (WS-BFR2, n = 3, 1 man and 2 women).

Training program
All subjects were instructed to walk at a self-selected, faster pace than usual for ≥30 min per day, ≥2-4 days per wk (≥4 days per wk in the WS group, ≥3 days per wk in the WS-BFR1 group, and ≥2 days per wk in the WS-BFR2 group), and climb ≥5 flights of stairs per day, ≥4 days per wk for 11 wk. Additionally, both the WS-BFR1 (once a wk) and WS-BFR2 (twice a wk) groups performed 20 min of treadmill walking with BFR at an exercise intensity of 70-85% of the age-predicted maximum heart rate (220 − age). During the BFR-walk session, nylon cuffs (105 mm wide, MT-870 Digital Tourniquet; Mizuho, Tokyo, Japan) were applied tightly at the most proximal portion of both legs. The target 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 mmHg and 50-55 cm = 120 mmHg. This is because arterial occlusion pressure is largely influenced by thigh circumference 22) . The cuff air pressure was released immediately after completing each session.

MT
MT was measured via B-mode ultrasound using a 5-18 MHz scanning head (Noblus; Aloka, Tokyo, Japan) at the anterior (AT) and posterior (PT) aspects of the right thigh at 50% of the thigh length between the lateral condyle of the femur and the greater trochanter, and at the anterior (AL) and posterior (PL) aspects of the right lower leg at 30% of the lower leg length between the lateral malleolus of the fibula and the lateral condyle of the tibia. Prior to all scans, subjects rested quietly in a seated position for at least 30 min. To avoid the influence of fluid shifts within the muscle, the measurements were performed around the same time. All measurements were performed by the same operator. The ultrasound measurements of MT were performed with subjects 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 before and after the training period. The scanning head, which was coated with a water-soluble transmission gel, was placed on each marked measurement site without depressing the dermal surface. The subcutaneous adipose tissue-muscle interface and the musclebone interface were identified on ultrasound images, and the distance between the two interfaces was recorded as the MT. The test-retest (inter-session) reliabilities of the MT measurements were calculated using an intraclass correlation coefficient, standard errors of measurement, and minimal difference. These values were previously determined in 10 young subjects in terms of the AT values, and were 0.999, 0.21 mm, and 0.58 mm, respectively.

10-m walking time
Walking performance was evaluated by timing each subject as they walked across a 10-m corridor on a hard-surfaced floor. The width of the corridor was set at 1-m. Subjects performed two timed trials and were encouraged to maintain a straight course. They were asked to walk down the corridor as fast as possible without running. Their times were measured using a digital stopwatch (LC058, Citizen, Tokyo, Japan), and the best time was used for the 10-m walking time.

Statistical analysis
All results are expressed as means with standard errors. The data of subjects whose adherence rate was ≥80% for both the walking and stair-climbing programs were used for analysis (WS: n = 8, 8 men; WS-BFR1: n = 8, 6 men and 2 women; WS-BFR2: n = 3, 1 man and 2 women). Statistical analysis featured two-way analysis of variance (ANOVA) with repeated measures [condition (WS, WS-BFR1, and WS-BFR2)× time (PRE and POST)].
All baseline values for all groups were compared using one-way ANOVA. Statistical significance was set at p < 0.05.

Results
The mean walking time per day, walking training frequency per wk, number of flights of stairs per day, and stair-climbing training frequency per wk in each group were as follows: 62.8 ± 7.8 min/day, 5.3 ± 0.4 days/wk, 11.2 ± 2.6 flights/day, and 5.1 ± 0.5 days/wk in the WS group; 48.7 ± 5.8 min/day, 4.4 ± 0.4 days/wk, 6.8 ± 0.6 flights/day, and 4.9 ± 0.4 days/wk in the WS-BFR1 group; and 52.0 ± 4.3 min/day, 4.3 ± 0.3 days/wk, 7.7 ± 1.3 flights/day, and 4.4 ± 0.7 days/wk in the WS-BFR2 group, respectively. The mean rates of perceived exertion during walking and stair-climbing training were 12.7 ± 0.5 and 13.4 ± 0.6 in the WS group, 11.7 ± 0.4 and 12.3 ± 0.2 in the WS-BFR1 group, and 10.8 ± 0.2 and 13.0 ± 0.6 in the WS-BFR2 group, respectively. There were no significant differences in these values among all groups. The adherence rates in BFR-walk training were 94% and 100% in the WS-BFR1 and WS-BFR2 groups, respectively. Applying BFR did not increase any relevant side effects such as subcutaneous hemorrhage, numbness, and cerebral anemia. There were no significant differences in body mass and the body mass index after a training program.
Table-1 shows the change of MT and walking performance before and after a 3-month training period. Two-way repeated measures ANOVA showed that the time effects were significant (p < 0.01) for MT of the PT (WS: 8.9%, WS-BFR1: 11.7%, WS-BFR2: 11.8%) and PL (WS: 3.0%, WS-BFR1: 1.1%, WS-BFR2: 6.6%). Moreover, MT of the AT and AL did not change. A significant interaction between the time and group was not observed for these MTs; i.e., there was no significant difference in these MT values among all the groups. For the 10-m walking time, the main effect of time was significant (p < 0.05, WS: 0.0%, WS-BFR1: -6.9%, WS-BFR2: -8.9%), but the interaction was not significant.

Discussion
The major findings of the present study were that 11 wk of walking and stair-climbing training increased MT of the PT and PL, and improved walking performance, whereas additional training effects induced by a BFR-walk once or twice per wk were not observed.
A number of studies have reported the effect of a walking program on body composition and aerobic capacity 23) , whereas only a few studies have investigated the influence of walk training on lower body muscle size. Kubo et al. demonstrated that MT of the PT increased by 7.6% after a 6-month walking training (45.0 ± 15.6 min/day and 5.4 ± 1.1 days/wk) 16) , whereas the present study showed that MT of the PT increased by 8.9% for the WS group following the 11-wk walking program (62.8 ± 7.8 min/day and 5.3 ± 0.4 days/wk). These results suggest that the hypertrophic effects of walking may reach a plateau after about 3 months into the program. In both studies, a significant increase in MT of the AT was not observed. Moreover, two previous studies have demonstrated no significant change in the thigh muscle cross-sectional area (CSA), which was not observed following both the 10-wk and 18-wk walking programs 21) 24) . Since the thigh muscle CSA includes both the AT and PT, site-specific muscle hypertrophy may be undetected.
Stair-climbing training was performed in the present study to work the quadriceps femoris muscles more than walking. One study demonstrated that the muscle activation levels of the quadriceps femoris muscles during ascending the stairs were 31.0 ± 10.9% and 28.7 ± 7.6%EMG (%electromyogram, the averaged integrated EMG during the action/that during maximal voluntary isometric contraction) in older men and women, respectively, whereas the activation levels of the triceps surae muscles were 46.9 ± 27.7% and 52.4 ± 34.5 %EMG, respectively 17) . In the present study, MT of the PL increased after the training program, but MT of the AT did not change, which may suggest that stair-climbing training provided enough stimuli to induce muscle hypertrophy to the PL, but not to the AT.
Muscle hypertrophy mainly results from exposure to both metabolic and mechanical stresses 19) . The application of BFR during exercise produces greater and/or faster metabolic fatigue, which would induce greater and/or faster muscle growth 19) . For example, our previous study showed that 20 min of a BFR-walk, 4 days per wk for 10 wk, increased thigh muscle CSA, whereas normal walking did not 21) . To develop a more practical training program (i. e., reduce the training frequency of a BFR-walk), the present study investigated whether a greater hypertrophic effect was observed in the WS-BFR1 and WS-BFR2 groups than in the WS group, but there was no significant difference in the hypertrophic effect among all the Values are presented as mean (± standard error). AT: anterior aspect of the thigh, PT: posterior aspect of the thigh, AL: anterior aspect of the lower leg, PL: posterior aspect of the lower leg Table-1 Changes in muscle thicknesses and walking performance after an 11-wk training program groups. The improvement rate in MT and walking performance tended to be higher in the WS-BFR-2 group than in the WS and WS-BFR1 groups. The training effect with body mass-based training (i.e., walking) depends on the ratio of body mass to lower body strength, which is lower in women than in men; thus, this may explain why a higher percentage of female subjects in the WS-BFR2 group had a higher increasing rate in the present study. Thus, future studies with a more robust experimental design need to investigate the training effect of a BFR-walk with less of a frequency (i.e., 1-2 days per wk).
In conclusion, walking and stair-climbing training can increase MT of the PT and PL, and improve walking performance in older subjects, whereas a BFR-walk once or twice a wk may not produce additional training effects.