宇宙航空環境医学 Vol. 56, No. 3, 39-48, 2019

Case Report

運動実践に伴う乳脂肪球皮膜サプリメント摂取が14日間のベッドレスト終了後における若年成人の骨格筋関連パラメータに及ぼす影響:パイロット研究

大須賀洋祐1,山崎 正志2,正田 純一2,長島 克弥2,鄭  松伊3,門根 秀樹4,落合 龍史5,大崎 紀子6,桂木 能久6,田中喜代次7

1東京都健康長寿医療センター研究所自立促進と精神保健研究チーム
2筑波大学医学医療系
3弘前大学医学部
4筑波大学附属病院つくば臨床医学研究開発機構未来医工融合研究センター
5花王株式会社生物科学研究所
6花王株式会社ヘルス&ウェルネス研究所
7筑波大学体育系

TEffects of exercise with milk-fat globule membrane supplementation after 14 days of bed rest on the skeletal muscle-related parameters in young adults:a pilot study

Toru Konishi1,2, Takuya Kurazumi1, Chiharu Takko1, Tomokazu Kato1, Yojiro Ogawa1, Ken-ichi Iwasaki1

1Research Team for Promoting Independence and Mental Halth, Tokyo Metropolitan Institute of Gerontology
2Faculty of Medicine, University of Tsukuba
3School of Medicine, Hirosaki University
4Center for Innovative Medicine and Engineering, University of Tsukuba Hospital
5Biological Science Research, Kao Corporation
6Health & Wellness Products Research, Kao Corporation
7Faculty of Health and Sport Sciences, University of Tsukuba

ABSTRACT
 Aim:This study aimed to provide preliminary data regarding the effects of exercising and milk-fat globule membrane (MFGM) supplementation on skeletal muscle-related parameters after bed rest. 
 Methods:Four young men (20.8±1.7 years) completed 14 days of bed rest and were randomized to the intervention group (exercise + MFGM, n=2) or the control group (exercise + placebo, n=2) for 28 days. Aerobic exercise and resistance exercise were performed on 2 nonconsecutive days per week during the 28-day recovery period. We assessed integrated values per sec during a performance of maximal muscle strength as the primary outcome measure using electromyography (EMG) and a dynamometer. The secondary outcome measures were muscle cross-sectional area, fat-free mass, agility, reactivity, cardiorespiratory fitness, and hematological parameters. All data were obtained at before and after bed rest, at 14-day (recovery 1) and at 28-day (recovery 2) after bed rest.
 Results:During the bed rest period, significant decrease in body weight and fat-free mass, and increase in homeostasis model assessment-insulin resistance, high-sensitivity C-reactive protein, insulin-like growth factor-1, and soluble receptor for advanced glycation end products were observed. There were no drastic differences in integrated values of EMG and agility;at recovery 1. However, the MFGM group exhibited a trend towards higher change in those parameters at recovery 2, compared to the control group.
 Conclusions:These parameters could be used to perform a randomized controlled trial that evaluates the effects of MFGM on EMG amplitude and agility after bed rest. However, to accurately evaluate the effectiveness of MFGM, prolonged MFGM supplementation may be needed to observe significant improvements.
 Registration:UMIN000020661 (February 1, 2016).

(Received:14 October, 2019 Accepted:16 April, 2020)

Key words:bed rest, muscle function, milk-fat globule membrane, exercise, sphingomyelin

1. INTRODUCTION
 Loss of skeletal muscle mass and function with increasing age is closely associated with adverse health outcomes, such as functional impairment and physical disability among older adults1). In addition, bed rest as a result of hospitalization causes significant loss of skeletal muscle function2). Therefore, developing countermeasures to prevent muscle function loss after bed rest is very important for promoting a physically independent lifestyle during old age.
 Previous studies have revealed that protein supplementation during bed rest may be a useful method for maintaining muscle strength3,4). Several other studies have suggested that exercising and supplementation using milk-fat globule membrane (MFGM) can help improve muscle function through neuromuscular development in mice and humans5-7). In this context, MFGM is a milk component that acts as an emulsifier and protects milk-fat globules from coalescence and enzymatic degradation. Moreover, MFGM is rich in bioactive products, such as phospholipids, sphingolipids, and membrane-specific phospholipids and sphingolipids8).
 However, the previous studies have only evaluated the effects of MFGM supplementation among individuals who had a normal lifestyle, and we are not aware of any studies that have evaluated whether exercising and MFGM supplementation can improve skeletal muscle-related parameters after bed rest. Thus, the present study aimed to obtain preliminary data regarding the effects of exercising with MFGM supplementation on electromyography (EMG), muscle mass, motor performance, and hematological parameters after bed rest.

2. METHODS
 1) Study design and participants  This double-blind randomized placebo-controlled trial was registered with the University Medical Information Network (UMIN000020661). The study protocol complied with the tenets of the Declaration of Helsinki, and was reviewed and approved by the Ethics Committees of the University of Tsukuba (Tai27-74, 13 October 2015), Tsukuba University Hospital (H27-178, 25 January 2016), and Japan Aerospace Exploration Agency (RinRiI Sho27-4-2, 4 February 2016). Participants were recruited from universities in a southern area of Ibaraki through recruitment posters, and four individuals volunteered to participate. After providing their written informed consent, the four participants were screened at the Tsukuba University Hospital using various medical evaluations and interviews. The inclusion and exclusion criteria are described in the University Medical Information Network registry (UMIN000020661). The four participants underwent baseline measurements as a screening test, and were then randomized by a non-investigator to the intervention group (2 participants; exercise and MFGM) or the control group (2 participants;exercise and placebo).
 2) Study protocol
 The study timeline and procedures are presented in Table 1. D-dimer test, urine tests, and ultrasonic evaluations were performed on day −15 and −7 to evaluate the safety of the participants (especially regarding whether they have developed lower-extremity deep vein thrombosis or urinary tract stones during the bed rest). During the recovery 1 phase (day 0 to 14), the participants stayed in the hospital and were treated with daily supplementation of MFGM or placebo tablets after breakfast, as is shown below. They also performed aerobic and resistance exercise training. The details of exercise training are described below. During the recovery 2 phase (days 14 to 28), the participants were discharged and returned home, but continued the same exercise program and daily supplementation of MFGM or placebo after breakfast at the hospital. Ingestion of coffee, tea, and/or alcohol was strictly prohibited during the study period.

Table 1 Study protocol and procedure
Screening
Day −19
Before bed rest
Day −15
Middle bed rest
Day −7
After bed rest
Day 0
Recovery 1 Recovery 2
Day 7 Day 14 Day 21 Day 28
Safety outcomes
 Medical history
 OH test
 Psychological test
 Resting ECG
 Vital signs
 Ultrasonic diagnosis
 Urine test
 D-dimer
Efficacy outcomes
 Muscle ECG
 Muscle strength
 Blood test
 CPX
 Agility
 Reactively
 CT
 DEXA

Note:OH, orthostatic hypotension;ECG, electrocardiogram;CPX, cardiopulmonary exercise test;CT, computed tomography;DEXA, dual-energy X-ray absorptiometry. ✓, data collection

 3) Bed rest
 All participants completed their 14 days of bed rest at the Tsukuba University Hospital. The participants performed all basic activities of daily living (e.g., eating, bathing, and toileting) in their beds, although the participants were allowed to move to a lavatory using a wheelchair if they experienced difficulty defecating in their bed. The nursing staff and investigators monitored the participants for safety and protocol compliance using their daily clinical and diary records.
 4) Diet, physical activity, and supplementation
 All participants received appropriate diets, based on the Harris-Benedict formula, at the hospital during the bed rest and recovery 1 periods. During the recovery 2 period, the participants were requested to maintain their habitual diet and physical activity. Each day after breakfast, the participants ingested 6 MFGM tablets (1 g MFGM/day:containing 37 mg sphingomyelin) or the placebo tablets (1 g whole milk powder/day) throughout the recovery period. 
 5) Exercise training
 Aerobic exercise training and resistance exercise training were performed on 2 nonconsecutive days per week at the hospital under the supervision of an expert trainer. The aerobic exercise training involved low-volume aerobic-type interval exercises training that were developed for adults with low-level physical fitness, and details regarding the exercise protocol have been previously published9). The resistance exercise training involved five resistance exercises (forward lunge, half squat, push-up, sit-up, and back extension). The participants completed 3 sets of 20 repetitions during the recovery 1 and 2 phases, with rest periods of 1 min between sets and 2 min between 5 resistance exercises.
 6) Primary outcome measures
 EMG’s integral value per sec was assessed using the isometric voluntary contractions of the quadriceps femoris muscles (rectus femoris, vastus lateralis, and vastus medialis) during maximum-strength knee extensions. Maximal isometric strength was measured using a dynamometer (System 3 Pro;Biodex Medical Systems Inc., NY, USA). The contractions were monitored using electromyography (TrignoTM Wireless Systems and Smart Sensors;DELSYS Inc., MA, USA). Primary outcomes were measured at Day −15, 0, 7, 14, 21, and 28.
 7) Secondary outcome measures
 Total cross-sectional area of whole thigh muscles in the mid-region was computed using magnetic resonance spectroscopy (Achieva;Philips Electronics Japan Ltd., Tokyo, Japan), and the data were analyzed using LCModel software (LA Systems Inc., Tokyo, Japan). Dual-energy X-ray absorption was used to estimate whole-body fat-free mass using a standard protocol (Hologic Horizon A, version 13.5.3.1). Agility was measured using a step counter (SW-4;Yagami Inc., Nagoya, Japan), and the participants were instructed to step as quickly as possible in the sitting position for 30 s. Reactivity was measured using unique equipment (SP-7;Yagami Inc., Nagoya, Japan) that randomly indicated 4 directions (forward, backward, right, or left), and the participants were instructed to take a step in the direction of each indication. The participants’ reaction times were averaged based on the results from 8 random tests. Cardiopulmonary exercise testing was performed to determine the participants’ peak oxygen uptake (VO2peak) values, and the methods for this test have been described in a previous study10). The participants rode an ergometer (75XL III;KONAMI Sports Life, Tokyo, Japan) and pedaled until physical exhaustion;after a 2-min warm-up at 30 W, the workload was increased by 30 W every min. The VO2 was measured using the breath-by-breath method and a computerized indirect calorimeter (Fitmate PRO;COSMED, Rome, Italy).
 Before the breakfast, fasting blood samples were collected from the antecubital vein of each participant. Total protein, albumin, creatine kinase, blood glucose, insulin, homeostasis model assessment-insulin resistance (fasting insulin × fasting blood glucose / 405), high-sensitivity C-reactive protein, tumor necrosis factor-alpha, interleukin-6, dehydroepiandrosterone sulfate, testosterone, insulin-like growth factor-1, 1,25-dihydroxyvitamin D, soluble receptor for advanced glycation end products, and malondialdehyde low-density lipoprotein were analyzed. Also, calcium levels in urine were analyzed to assess a risk of ureteral stone onset (data are not shown).
 Muscle cross-sectional area and fat-free mass were measured at Day −15, 0, and 28, and the other secondary outcomes were measured at Day −15, 0, 14, and 28.
 8) Statistical analysis
 Paired t-tests were used to evaluate the differences in the baseline and post-bed rest measurements from the four participants. All analyses were performed using SPSS software (version 22.0;SPSS Inc., Chicago, IL, USA). A P-value of <0.05 was considered significant. All data were reported as mean±standard deviation.

3. RESULTS AND COMMENTS
 The participants’ average age was 20.8±1.7 years, and all four participants successfully completed the study. No severe adverse events were observed during the study period, although mild headache and backache were reported during the early bed rest period (n=1). The change patterns in EMG, muscle mass, motor performance, and blood parameters are shown in Tables 2 to 5.
 During the bed rest period, significant decrease in body weight and fat-free mass, and increase in homeostasis model assessment-insulin resistance, high-sensitivity C-reactive protein insulin-like growth factor-1, and soluble receptor for advanced glycation end products were observed. This suggests that these parameters may be useful for assessing the influence of bed rest. However, the other variables did not exhibit any noticeable trends. Although no remarkable difference in change in EMG parameters and agility between the two groups during the recovery 1 period (change from day 0 to 14), noticeable increases in EMG parameters and agility were observed in the MFGM group during the recovery 2 period (change from day 14 to 28). These results may suggest that a prolonged period of MFGM supplementation is needed to affect EMG amplitude and agility. However, this relationship is speculative, as we could not collect sufficient data to perform appropriate statistical analyses of the effects of MFGM. Furthermore, our results are limited by the small sample size, and a larger randomized controlled trial with a prolonged follow-up is needed to evaluate the effects of MFGM. Nevertheless, to the best of our knowledge, this is the first study to evaluate the effects of exercising with MFGM supplementation on action potential and mass, motor performance, and hematological parameters after bed rest. Therefore, our findings may be useful for designing a larger randomized controlled trial in the future. 

Table 2 Change pattern in morphology
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1 Recovery 2
Day 7 Day 14 Day 21 Day 28
Body weight, kg
 All, n=4 74.1±8.3 72.5±8.2* n/a 72.9±7.3 n/a 72.6±7.7
  MFGM, n=2 71.0±11.7 69.2±10.7 n/a 69.4±8.8 n/a 68.9±9.0
   Participant A 62.7 61.6 n/a 63.2 n/a 62.5
   Participant B 79.2 76.7 n/a 75.6 n/a 75.2
  Placebo, n=2 77.3±5.7 75.9±6.4 n/a 76.4±5.7 n/a 76.3±6.5
   Participant C 81.3 80.4 n/a 80.4 n/a 80.9
   Participant D 73.3 71.3 n/a 72.4 n/a 71.7
Total muscle cross-sectional area, cm2
 All, n=4 170.5±16.3 168.9±10.2 n/a n/a n/a 173.7±9.0
  MFGM, n=2 176.6±24.2 170.9±17.1 n/a n/a n/a 171.4±14.9
   Participant A 159.5 158.8 n/a n/a n/a 160.9
   Participant B 193.7 183.0 n/a n/a n/a 181.9
  Placebo, n=2 164.4±7.8 166.6±15.8 n/a n/a n/a 175.9±0.8
   Participant C 158.9 167.8 n/a n/a n/a 176.0
   Participant D 170.0 165.5 n/a n/a n/a 175.9
Fat-free mass, kg
 All, n=4 56.5±5.7 55.5±5.7* n/a n/a n/a 56.6±5.1
  MFGM, n=2 54.6±8.9 53.2±8.4 n/a n/a n/a 53.9±6.3
   Participant A 48.3 47.3 n/a n/a n/a 49.5
   Participant B 60.8 59.2 n/a n/a n/a 58.3
  Placebo, n=2 58.4±1.9 57.8±2.0 n/a n/a n/a 59.4±3.2
   Participant C 59.8 59.2 n/a n/a n/a 61.6
   Participant D 57.1 56.4 n/a n/a n/a 57.1

Note:n/a, not available, group data are shown as means±and standard deviations.
*:significant changes between the before and after bed rest measurements at P <0.05.



Table 3 Change pattern in electromyography and muscle strength
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1 Recovery 2
Day 7 Day 14 Day 21 Day 28
Vastus medialis, V/s
 All, n=4 0.27±0.07 0.19±0.03 0.21±0.04 0.25±0.03 0.23±0.06 0.26±0.04
  MFGM, n=2 0.31±0.08 0.21±0.01 0.21±0.04 0.27±0.04 0.23±0.11 0.30±0.01
   Participant A 0.25 0.20 0.18 0.24 0.15 0.29
   Participant B 0.36 0.22 0.24 0.29 0.30 0.31
  Placebo, n=2 0.23±0.06 0.17±0.01 0.20±0.04 0.24±0.04 0.24±0.03 0.23±0.01
   Participant C 0.18 0.16 0.17 0.21 0.22 0.22
   Participant D 0.27 0.17 0.23 0.26 0.26 0.23
Rectus femoris, V/s
 All, n=4 0.19±0.07 0.16±0.06 0.18±0.09 0.17±0.09 0.16±0.08 0.17±0.05
  MFGM, n=2 0.17±0.02 0.12±0.01 0.16±0.06 0.13±0.01 0.13±0.01 0.15±0.01
   Participant A 0.18 0.12 0.20 0.14 0.13 0.16
   Participant B 0.15 0.11 0.11 0.12 0.12 0.14
  Placebo, n=2 0.21±0.11 0.21±0.06 0.21±0.13 0.22±0.13 0.19±0.11 0.18±0.07
   Participant C 0.13 0.16 0.11 0.12 0.11 0.13
   Participant D 0.28 0.25 0.30 0.31 0.27 0.23
Vastus lateralis, V/s
 All, n=4 0.25±0.07 0.17±0.03 0.21±0.13 0.23±0.15 0.23±0.15 0.25±0.10
  MFGM, n=2 0.23±0.01 0.15±0.02 0.13±0.01 0.13±0.00 0.12±0.01 0.18±0.01
   Participant A 0.24 0.16 0.12 0.13 0.11 0.19
   Participant B 0.22 0.13 0.13 0.13 0.13 0.17
  Placebo, n=2 0.28±0.12 0.20±0.01 0.29±0.15 0.33±0.16 0.33±0.16 0.32±0.12
   Participant C 0.19 0.19 0.18 0.22 0.22 0.23
   Participant D 0.36 0.21 0.39 0.44 0.44 0.40
Isometric strength, Nm/kg
 All, n=4 223.6±47.7 165.5±46.6 153.8±40.2 175.7±46.8 191.8±68.2 211.4±55.0
  MFGM, n=2 214.5±79.6 140.0±23.4 121.4±14.2 138.8±33.1 135.6±31.9 167.7±21.3
   Participant A 158.2 123.4 111.3 115.4 113 152.6
   Participant B 270.8 156.5 131.4 162.2 158.1 182.7
  Placebo, n=2 232.7±12.4 191.1±57.8 186.2±21.0 212.7±4.9 248.0±17.6 255.1±31.0
   Participant C 223.9 232.0 201.0 216.1 260.4 277.0
   Participant D 241.5 150.2 171.3 209.2 235.5 233.2

Note:n/a, not available, group data are shown as means±and standard deviations.
*:significant changes between the before and after bed rest measurements at P <0.05.



Table 4 Change pattern in physical performance
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1 Recovery 2
Day 7 Day 14 Day 21 Day 28
Agility, times/15 s
 All, n=4 147.3±12.9 152.5±7.2 n/a 151.5±15.9 n/a 156.1±14.7
  MFGM, n=2 157.8±0.4 158.5±0.7 n/a 150.0±25.5 n/a 166.8±8.1
   Participant A 158 158 n/a 168 n/a 173
   Participant B 158 159 n/a 132 n/a 161
  Placebo, n=2 136.8±7.4 146.5±3.5 n/a 153.0±9.9 n/a 145.5±11.3
   Participant C 132 149 n/a 160 n/a 154
   Participant D 142 144 n/a 146 n/a 138
Reactivity, s
 All, n=4 0.89±0.14 0.90±0.09 n/a 0.85±0.12 n/a 0.83±0.11
  MFGM, n=2 0.79±0.13 0.83±0.05 n/a 0.78±0.15 n/a 0.77±0.14
   Participant A 0.70 0.79 n/a 0.68 n/a 0.67
   Participant B 0.88 0.87 n/a 0.89 n/a 0.87
  Placebo, n=2 0.99±0.07 0.97±0.03 n/a 0.91±0.05 n/a 0.90±0.01
   Participant C 1.04 0.99 n/a 0.95 n/a 0.89
   Participant D 0.94 0.96 n/a 0.88 n/a 0.90
Peak Oxygen Uptake, mL/min/kg
 All, n=4 45.5±7.3 43.9±3.8 n/a 47.5±6.8 n/a 46.8±7.5
  MFGM, n=2 51.7±1.9 50.0±4.7 n/a 52.8±1.8 n/a n/a
   Participant A 53.0 49.3 n/a 54.1 n/a 54.7
   Participant B 50.3 42.6 n/a 51.5 n/a n/a
  Placebo, n=2 39.3±1.3 41.9±2.3 n/a 42.2±4.7 n/a 42.9±4.3
   Participant C 40.2 43.5 n/a 45.5 n/a 45.9
   Participant D 38.4 40.3 n/a 38.9 n/a 39.8

Note:n/a, not available, group data are shown as means±and standard deviations.
*:significant changes between the before and after bed rest measurements at P <0.05.



Table 5a Change pattern in blood parameters
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1
Day 14
Recovery 2
Day 28
Total protein, g/dL
 All, n=4 7.4±0.4 7.4±0.4 7.3±0.3 7.3±0.4
  MFGM, n=2 7.1±0.2 7.2±0.5 7.1±0.0 7.0±0.3
   Participant A 7.2 6.8 7.1 6.8
   Participant B 6.9 7.5 7.1 7.2
  Placebo, n=2 7.7±0.3 7.7±0.2 7.6±0.1 7.5±0.3
   Participant C 7.9 7.8 7.5 7.7
   Participant D 7.5 7.5 7.6 7.3
Albumin, g/dL
 All, n=4 4.6±0.2 4.7±0.1 4.7±0.2 4.7±0.2
  MFGM, n=2 4.6±0.4 4.7±0.1 4.6±0.3 4.6±0.1
   Participant A 4.8 4.7 4.8 4.6
   Participant B 4.3 4.6 4.4 4.5
  Placebo, n=2 4.7±0.1 4.8±0.1 4.7±0.0 4.8±0.2
   Participant C 4.8 4.8 4.7 4.9
   Participant D 4.6 4.7 4.7 4.6
Creatine kinase, IU/L
 All, n=4 205.0±117.0 71.0±30.7 138.8±77.9 133.5±54.4
  MFGM, n=2 142.0±8.5 55.0±15.6 74.0±31.1 105.5±37.5
   Participant A 148.0 44.0 52.0 79.0
   Participant B 136.0 66.0 96.0 132.0
  Placebo, n=2 268.0±158.4 87.0±39.6 203.5±21.9 161.5±65.8
   Participant C 156.0 59.0 188.0 115.0
   Participant D 380.0 115.0 219.0 208.0


Table 5b Change pattern in blood parameters
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1
Day 14
Recovery 2
Day 28
Blood glucose, mg/dL
 All, n=4 91.8±9.1 84.5±2.9 84.8±4.0 89.0±1.2
  MFGM, n=2 84.0±1.4 84.5±3.5 86.0±1.4 89.0±1.4
   Participant A 85.0 87.0 85.0 90.0
   Participant B 83.0 82.0 87.0 88.0
  Placebo, n=2 99.5±2.1 84.5±3.5 83.5±6.4 89.0±1.4
   Participant C 98.0 87.0 88.0 90.0
   Participant D 101.0 82.0 79.0 88.0
Insulin, μU/mL
 All, n=4 7.8±1.9 10.1±2.8 10.5±2.6 9.3±3.9
  MFGM, n=2 7.4±2.0 8.4±2.1 10.0±1.6 11.7±3.9
   Participant A 8.8 9.9 11.1 14.4
   Participant B 6.0 6.9 8.8 8.9
  Placebo, n=2 8.2±2.6 11.8±2.8 11.0±4.0 6.9±2.6
   Participant C 10.0 13.7 8.1 5.0
   Participant D 6.3 9.8 13.8 8.7
Homeostatic model assessment of insulin resistance
 All, n=4 1.8±0.5 2.1±0.6* 2.2±0.4 2.0±0.9
  MFGM, n=2 1.5±0.4 1.8±0.5 2.1±0.3 2.6±0.9
   Participant A 1.9 2.1 2.3 3.2
   Participant B 1.2 1.4 1.9 1.9
  Placebo, n=2 2.0±0.6 2.5±0.6 2.3±0.6 1.5±0.6
   Participant C 2.4 2.9 1.8 1.1
   Participant D 1.6 2.0 2.7 1.9


Table 5c Change pattern in blood parameters
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1
Day 14
Recovery 2
Day 28
High sensitivity C-reactive protein, mg/dL
 All, n=4 0.016±0.009 0.023±0.007* 0.041±0.030 0.036±0.019
  MFGM, n=2 0.017±0.020 0.022±0.013 0.062±0.035 0.042±0.026
   Participant A 0.028 0.031 0.037 0.023
   Participant B 0.006 0.013 0.086 0.060
  Placebo, n=2 0.016±0.002 0.023±0.001 0.022±0.002 0.032±0.017
   Participant C 0.017 0.024 0.023 0.044
   Participant D 0.014 0.022 0.020 0.020
Tumor necrosis factor-alpha, pg/mL
 All, n=4 5.5±2.7 2.1±0.4 2.0±0.2 1.6±0.2
  MFGM, n=2 5.2±1.7 2.2±0.4 1.9±0.0 1.5±0.2
   Participant A 4.0 1.9 1.9 1.4
   Participant B 6.3 2.5 1.9 1.7
  Placebo, n=2 5.6±4.3 2.1±0.5 2.0±0.4 1.6±0.1
   Participant C 2.8 1.7 1.9 1.7
   Participant D 8.9 2.4 2.2 1.5
Interleukin-6, pg/mL
 All, n=4 3.4±2.9 1.1±0.3 0.9±0.4 2.4±2.4
  MFGM, n=2 2.5±1.5 0.9±0.4 1.0±0.6 1.7±1.7
   Participant A 1.4 0.6 0.6 0.5
   Participant B 3.5 1.2 1.5 2.9
  Placebo, n=2 4.3±4.4 1.2±0.3 0.6±0.1 3.1±3.5
   Participant C 1.2 1.4 0.7 5.6
   Participant D 7.5 1.0 0.6 0.6


Table 5d. Change pattern in blood parameters
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1
Day 14
Recovery 2
Day 28
Dehydroepiandrosterone sulfate, μg/dL
 All, n=4 391.5±99.9 288.8±70.5 337.5±88.7 314.5±55.8
  MFGM, n=2 400.0±19.8 310.0±106.1 333.5±99.7 316.0±60.8
   Participant A 414 235 263 273
   Participant B 386 385 404 359
  Placebo, n=2 383.0±171.1 267.5±43.1 341.5±116.7 313.0±75.0
   Participant C 504 298 424 366
   Participant D 262 237 259 260
Testosterone, ng/mL
 All, n=4 4.98±1.46 5.86±1.80 5.51±1.60 5.62±1.43
  MFGM, n=2 5.90±0.16 6.01±1.56 6.21±1.29 5.64±0.45
   Participant A 5.79 7.1 7.12 6.0
   Participant B 6.01 4.9 5.3 5.3
  Placebo, n=2 4.07±1.73 5.71±2.69 4.81±2.00 5.60±2.44
   Participant C 5.29 7.6 6.22 7.3
   Participant D 2.84 3.8 3.39 3.9
Insulin-like growth factor-1, ng/mL
 All, n=4 186.3±16.5 301.3±59.9* 271.0±52.4 220.3±31.4
  MFGM, n=2 173.0±9.9 268.5±29.0 235.0±42.4 198.5±3.5
   Participant A 180.0 289.0 265.0 201.0
   Participant B 166.0 248.0 205.0 196.0
  Placebo, n=2 199.5±3.5 334.0±75.0 307.0±35.4 242.0±32.5
   Participant C 197.0 281.0 282.0 219.0
   Participant D 202.0 387.0 332.0 265.0


Table 5e Change pattern in blood parameters
Before bed rest
Day −14
After bed rest
Day 0
Recovery 1
Day 14
Recovery 2
Day 28
1,25-dihydroxyvitamin D3, pg/mL
 All, n=4 43.6±10.5 32.0±5.4 34.6±10.8 38.1±12.1
  MFGM, n=2 37.1±12.7 32.1±7.1 31.3±14.3 38.9±19.2
   Participant A 28.1 27.1 21.2 25.3
   Participant B 46.1 37.2 41.4 52.5
  Placebo, n=2 50.1±1.1 31.9±6.2 37.9±10.0 37.2±8.3
   Participant C 50.9 36.2 45 43.1
   Participant D 49.3 27.5 30.8 31.3
Soluble receptor for advanced glycation end products, pg/mL
 All, n=4 1,743±712 2,014±627* 1,835±321 1,739±658
  MFGM, n=2 1,846.5±1,068.4 2,123.0±963.1 1,936.0±509.1 1,960.0±913.6
   Participant A 1,091 1,442 1,576 1,314
   Participant B 2,602 2,804 2,296 2,606
  Placebo, n=2 1,639.5±580.5 1,905.0±452.5 1,733.5±91.2 1,519.5±521.1
   Participant C 1,229 1,585 1,669 1,151
   Participant D 2,050 2,225 1,798 1,888
Malondialdehyde low-density lipoprotein, U/L
 All, n=4 90±13 116±26 133±20 84±24
  MFGM, n=2 88.0±21.2 128.5±21.9 136.0±1.4 91.0±33.9
   Participant A 73 113 137 67
   Participant B 103 144 135 115
  Placebo, n=2 92.5±7.8 103.0±29.7 129.5±33.2 77.5±19.1
   Participant C 98 82 106 64
   Participant D 87 124 153 91

ACKNOWLEDGEMENTS
 The authors thank Dr. Hiroshi Ohshima (Japan Aerospace Exploration Agency) for his great contribution in conducting this study. The authors also acknowledge the excellent work and dedication of the staff at the Tsukuba University Hospital and Japan Aerospace Exploration Agency.

CONFLICTS OF INTEREST
 This study was supported by a grant from Kao Corporation. Yoshihisa Katsuragi, Noriko Osaki, and Ryuji Ochiai are employed by Kao Corporation. These authors assisted in the study’s conception and data interpretation, but had no role in the study’s design, data acquisition, or data analysis. 

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Address for correspondence:
 Research Team for Promoting Independence and Mental Health, Tokyo Metropolitan Institute of Gerontology
 Yosuke Osuka
 Tel:03-3964-3241
 E-mail:osuka@tmig.or.jp