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ORIGINAL
Effects of daily 1,000-IU vitamin D-fortified milk intake onskeletal muscle mass, power, physical function andnutrition status in JapaneseYasushi Matsuura1, Teruhiro Morishita1, Michiko Sato1, Nami Sumida1, Takafumi Katayama2, Rie Tsutsumi3,
Hiroshi Sakaue3, Yutaka Taketani4, Koichi Sairyo5, Akihiko Kawaura1, and Eiji Takeda1
1
Kenshokai Gakuen College for Health and Welfare, Tokushima, Japan, 2Department of Statistics and Computer Science, College of Nursing
Art and Science, University of Hyogo, Akashi, Japan, 3Department of Nutrition and Metabolism, Institute of Biomedical Sciences, Tokushima
University Graduate School, Tokushima, Japan, 4Department of Clinical Nutrition and Food Management, Institute of Biomedical Sciences,
Tokushima University Graduate School, Tokushima, Japan, 5Department of Orthopedics, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan

Abstract : An intervention study was conducted to investigate the effects of daily 1,000-IU vitamin D-fortified
milk intake on skeletal muscle mass, power, physical function and nutrition status in 26 healthy people and 8
older adults living in a nursing home. The serum 25-hydroxyvitamin D [25(OH)D] level was 13.4 ± 0.8 ng / mL and it
markedly increased to 29.6 ± 0.9 ng / mL after daily 1000-IU vitamin D-fortified milk intake for 6 months. Handgrip
strength (kg) also significantly increased in the 21-50 years and total groups, and male subjects, and the timed up
and go test signiﬁcantly improved in the 21-50 years and total groups, and female subjects after 6-month vitamin
D intake. However, there were no significant differences between baseline and post-treatment in the Barthel
Index (BI), walking speed (m / sec) or skeletal muscle mass (kg, % of BW, kg / m2). Therefore, the present study
suggested that vitamin D-fortified milk intake is effective at improving muscle strength and physical function in
Japanese, although further studies are needed, particularly for older adults. J. Med. Invest. 68 : 249-255, August,2021
Keywords : Vitamin D supplementation, Serum 25-hydroxyvitamin D level, Skeletal muscle mass, Muscle strength

INTRODUCTION
Sarcopenia is the most common cause of musculoskeletal
disability in the elderly. It is characterized by the gradualage-related loss of muscle mass, muscle strength and physicalperformance, which leads to reduced mobility and an increased
risk of falls, and is associated with premature death (1-3). In
our previous studies, an age-related decrease in total-bodyskeletal muscle mass based on 24-hour creatinine excretion andage-related loss of muscle power and function were observed ina Japanese population including nursing home residents (4-6).
Furthermore, a low vitamin D level is positively associated with
reduced skeletal muscle mass, muscle function and strength. As
vitamin D deﬁciency is prevalent and is associated with poor
physical performance in older populations (7-9), correcting thisdeﬁciency may be important for protecting muscle function withage (10).
The beneficial effects of vitamin D supplementation on muscle
mass, muscle strength and physical performance have been reported in older people with a low level of serum vitamin D (11-13).
However, some reports of vitamin D supplementation noted no
improvement in muscle strength or physical function (14-16). A
daily dose vitamin D of 800-2000 IU was recommended to reacha serum 25(OH)D level of 75 nmol / L in study participants witha low vitamin D status (17). Therefore, the effects of daily 1,000IU vitamin D-fortified milk intake on skeletal muscle mass,
Received for publication December 1, 2020 ; accepted April 8, 2021.
Address correspondence and reprint requests to Eiji Takeda, Kenshokai Gakuen College for Health and Welfare, 369-1 Higashitakawa, Tenma, Kokufu-cho, Tokushima, 779-3105, Japan andFax : +81-88-642-9227.

power, physical function and nutrition status were investigatedin a population of young to old Japanese subjects, including older
nursing home residents.

MATERIALS AND METHODS
Subjects

This was an intervention study that investigated the effects ofdaily 1,000-IU vitamin D-fortified milk intake in 200 ml of cow
milk for 6 months on skeletal muscle mass, physical function, intestinal nutrient absorption rate, and vitamin and mineral status. The milk was manufactured by Meiji Co., Ltd., Tokyo, Japanand contains 137 kcal, 9.9 g of carbohydrates, 6.8 g of protein, 7.8
g of fat, 227 mg of calcium and 1000 IU of vitamin D. The numbers of male and female subjects in the age groups of 21-50 years,
51-75 years and 76 years or older were 11 and 6, 5 and 8, and 0
and 4, respectively. Among them, male and female residents innursing homes accounted for 4 and 0 in the 51-75 years groupand 0 and 4 in the 76 years or older group, respectively. The other26 subjects aged between 21-75 years old were healthy registeredcare workers or physical and occupational therapists at nursing
homes, or teaching and administration staff at Kenshokai Gakuen College for Health and Welfare. None of the healthy subjects were engaged in high levels of exercise training or taking
any medications just before or during the study. Routine bloodstudies, including electrolytes, liver tests and hematologicalindexes, confirmed the health status of each subject before entry
into the study. Height and weight measurements were performed
with the participants wearing light clothing and no shoes. The
body mass index (BMI) was calculated as weight (kg) divided by
the square of height (m).

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Y. Matsuura, et al. Vitamin D supplementation and skeletal muscle

Clinical laboratory tests
Blood samples were collected from subjects who fasted formore than 8 hours overnight, immediately refrigerated, transported in cold storage to the SRL Laboratory in Tokyo, andanalyzed within 24 hours. Serum levels of sodium, potassium,chloride, calcium, phosphorus, zinc, iron, cupper, folic acid, b-carotene, 25-hydroxyvitamin D [25(OH)D] and intact parathyroid
hormone (PTH) were measured. Serum 25(OH)D levels, as anindicator of the vitamin D status, were measured by electro-chemiluminescent immunoassay (ECLIA) as previously reported(5, 6). There is a general consensus that a serum 25(OH)D
concentration of < 20 ng / ml, 20 to < 30 ng / mL and ≥ 30 ng / mL
indicates vitamin D deficiency (VDD), insufficiency (VDI), andsufficiency (VDS), respectively (18).

Measurement of skeletal muscle mass (SMM)
Each subject collected two 24-hour urine samples at inclusion
on two consecutive days. Creatinine excretion was calculated
as the mean of the two 24-hour urine samples. SMM was calculated from the 24-hour urinary creatinine amount based on
this equation (SMM (kg) = 21.8 × Cr (g / day)) (19). The SMM
index (SMMI) was calculated as weight (kg) divided by squareof height (m). SMM (% of body weight (kg)) was calculated asweight (%) divided by body weight (kg). Creatinine height index(CHI) was calculated from the following formula :CHI = 24-hour urine creatinine excretion (mg) / expected 24hour urine creatinine excretion (23 mg / kg in males and 18
mg / kg in females) × 100.
The estimated nitrogen balance was calculated as the difference between total nitrogen intake and total nitrogen output
in the urine. Nitrogen intake was calculated from the nitrogenprovided in food for each subject. The following formula was usedin calculating the nitrogen balance : Nitrogen balance = 24-hour
protein uptake / 6.25-(24-hour nitrogen excretion / 0.8). Putative
intestinal absorption rates of nitrogen, sodium, calcium andphosphorus balance were calculated as the urinary excretion
rate (%) among the amount of oral intake from food.

Assessment of physical performance
Physical performance was evaluated through several physicaltests such as the timed up and go (TUG) test (sec), walking speed(m / sec), handgrip strength (kg) and Barthel Index (BI). Muscle
strength was assessed as handgrip strength using a dynamometer (Takei Scientific, Tokyo, Japan). Both hands were measured
twice and the maximum value of either hand was analyzed. For
the TUG test, individuals were asked to rise from a standard
chair, walk to a marker 3 m away, turn around, walk back andsit down again (20). The BI is a 10-item measurement tool ofbasic activities of daily living (ADL) (21). It is used in clinicalpractice to inform rehabilitation and care planning, and in research both to describe outcomes and as a case-mix adjuster (22).

Statistical analysis
Using BellCurve for Excel (version 3.20) (Social Survey Research Information Co., Ltd., Tokyo, Japan), the Wilcoxon signed
rank test was performed to assess changes in skeletal muscle
function, muscle mass and intestinal nutrition absorption rates
after vitamin D supplementation, and the association between
serum 25(OH)D levels and skeletal muscle mass and bodyfunctions.
Data are expressed as the mean ± SE of male and female subjects in different age groups.

Ethical considerations
Ethics approval was obtained from the clinical research ethics

committee at Tokushima University Hospital (approval number
384). Informed consent to participate in the study was also received from participants or from an authorized surrogate. This
trial was registered as UMIN000038105.

RESULTS

1. Effects of vitamin D fortified milk intake in different age groups(Table 1)
At baseline, the serum 25(OH)D level in the 34 male and female subjects was 13.4 ± 0.8 ng / mL. Thirty participants (88.2%)
had VDD and 4 (11.8%) had VDI. After 1,000-IU vitamin D-fortified milk intake per day for 6 months, there was a signiﬁcant
increase in the serum 25(OH)D level to 29.6 ± 0.9 ng / mL. In different age groups, the serum 25(OH)D level markedly improvedin the 21-50 years (n = 17), 51-75 years (n = 13), 21-75 years(n = 30) and total (n = 34) groups, but not in the 76 years or older(n = 4) group. Only two participants (5.8%) had VDD, 16 (47.1%)
had VDI, and 16 (47.1%) had VDS. After vitamin D-fortified
milk intake for 6 months, serum calcium and phosphorus levels
increased, and PTH level decreased in these subjects.
Handgrip strength and TUG signiﬁcantly improved after
vitamin D supplementation in the 21-50 years and total groups,
but there were no signiﬁcant differences after treatment in BI,
walking speed (sec / m) or skeletal muscle mass (kg, % of BW,
kg / m2). A significant increase in skeletal muscle mass (kg) was
observed only in the 51-75 years group. Regarding the intestinal
absorption rate after vitamin D-fortified milk intake, those of
nitrogen and calcium decreased, and that of phosphorus increased in the 21-50 years and total groups, but that of sodium
was unaffected.

2. Effects of vitamin D fortified milk intake in male and female
groups (Table 2)
At baseline, the serum 25(OH)D level in 16 male and 18
female subjects was 14.6 ± 1.5 ng / mL and 12.4 ± 0.8 ng / mL,
respectively. Thirteen (81%) and 3 (19%) male, and 17 (94%) and
1 (6%) female subjects had VDD and VDI, respectively. After
1,000-IU vitamin D-fortified milk intake per day for six months,
the serum 25(OH)D level significantly increased to 30.0 ± 1.2
ng / mL (9 (56%) VDI and 7 (44%) VDS) in male subjects and29.2 ± 1.4 ng / mL (2 (11%) VDD, 7 (39%) VDI and 9 (50%) VDS)
in female subjects. After vitamin D-fortified milk intake for6 months, serum calcium and phosphorus levels increased infemale subjects.
Although skeletal muscle mass was unaffected by vitamin
D supplementation in both male and female groups, handgrip
strength in males and TUG in females signiﬁcantly improved.
Regarding the intestinal absorption rate after vitamin D-fortified milk intake, that of nitrogen decreased in males and that ofphosphorus increased in females.

3. Association of 25(OH)D with skeletal muscle mass, skeletal muscle function and predicted intestinal absorption rate
A positive correlation between serum 25(OH)D levels and theBI, grip-hand strength, and nitrogen and calcium absorption
rates were observed at baseline (r = -0.32, p < 0.001). After
vitamin D-fortified milk intake, the serum 25(OH)D level was
also positively correlated with skeletal muscle mass (kg, kg / m2)(r = -0.20, p = 0.007).

DISCUSSION
Aging results in a decrease in muscle strength and this is

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The Journal of Medical Investigation Vol. 68 August 2021
Table 1. Effects of vitamin D-fortified milk intake in different age groups
21-50 years
Baseline
MeanSE

51-75 years

6 months
Mean

BaselineSE

Mean

76 years〜

6 months

SE

Mean

SE

Baseline
Mean

SE

Total

6 months
Mean

SE

BaselineMean

6 monthsSE

Age (years)

37.8 ± 1.5

60.3 ± 2.1

86.5 ± 2.7

52.1 ± 3.1

n (male / female)17(11/6)

13(5/8)4(0/4)

34(16/18)

Mean

SE

SMM
SMM (kg)

28.1 ± 2.4

29.2 ± 2.2

20.7 ± 1.8

23.8 ± 3.2 *8.9 ± 1.9

6.0 ± 1.5

23.0 ± 1.7

24.4 ± 2.0

SMM (% of body weight)

42.9 ± 3.5

44.2 ± 3.5

35.1 ± 3.5

39.4 ± 5.6

18.4 ± 3.1

13.0 ± 3.4

37.0 ± 2.6

38.7 ± 3.2

SMM (kg / m2)

10.7 ± 0.9

10.9 ± 0.9

8.2 ± 0.8

8.9 ± 1.2

4.1 ± 0.9

2.7 ± 0.7

9.0 ± 0.7

9.2 ± 0.8

Creatinine height index (%)

92.4 ± 7.3

93.7 ± 6.9

82.4 ± 7.2

93.9 ± 13.4

46.9 ± 8.0

33.1 ± 8.7

83.2 ± 5.2

86.7 ± 7.0

Timed up and go test (sec)

6.3 ± 0.4

5.3 ± 0.1 *9.8 ± 3.7

11.7 ± 4.4--

8.3 ± 1.5

8.4 ± 1.8 **Walking speed (m / sec)

3.3 ± 0.2

3.2 ± 0.2

4.1 ± 0.9

4.3 ± 1.1--

3.8 ± 0.4

3.7 ± 0.4

Handgrip strength (kg)40.7 ± 2.7

42.3 ± 2.8 **23.6 ± 3.3

25.1 ± 2.6

8.4 ± 1.6

6.4 ± 3.2

31.0 ± 2.7

32.3 ± 2.7 *Barthel index

100.0 ± 0.0

100.0 ± 0.0

84.2 ± 7.9

83.5 ± 7.9

31.3 ± 13.1

30.0 ± 13.7

85.9 ± 4.9

85.4 ± 5.0
50.0 ± 3.1 ***SMF

Predicted intestinal absorption rate
Nitrogen (%)

76.6 ± 5.1

56.7 ± 3.2 **65.2 ± 4.5

50.3 ± 4.1

36.5 ± 11.8

20.3 ± 9.2

67.6 ± 3.9

Sodium (%)

86.4 ± 6.8

77.3 ± 4.7

82.3 ± 9.7

86.0 ± 9.7

70.4 ± 10.0

80.8 ± 22.0

83.0 ± 5.1

81.1 ± 4.9

Calcium (%)

15.2 ± 1.9

11.7 ± 1.3 *22.4 ± 3.8

20.8 ± 2.8

6.8 ± 3.3

4.0 ± 1.7

17.0 ± 1.9

Phosphorus (%)

59.4 ± 4.3

70.5 ± 4.6 *14.3 ± 1.6 **51.9 ± 4.4

61.5 ± 7.3

22.8 ± 6.0

23.3 ± 5.5

52.2 ± 3.4

61.5 ± 4.4 ***Calcium (mg / dL)

9.3 ± 0.1

9.4 ± 0.1 *9.2 ± 0.1

9.4 ± 0.1 **8.6 ± 0.1

8.8 ± 0.1

9.2 ± 0.1

9.3 ± 0.1 ***Phosphorus (mg / dL)

3.6 ± 0.1

3.8 ± 0.1

3.5 ± 0.1

3.6 ± 0.1

4.2 ± 0.1

3.5 ± 0.1

25(OH)D (ng / mL)

14.5 ± 1.0

29.7 ± 1.3 ***12.8 ± 1.6

28.1 ± 1.5 **10.8 ± 0.5

33.9 ± 0.4

13.4 ± 0.8

Intact PTH (pg / mL)

41.7 ± 3.1

37.5 ± 4.0

40.5 ± 6.3

34.7 ± 2.5

41.5 ± 4.3

34.3 ± 6.5

41.2 ± 2.8

Concentration in serum
3.8 ± 0.1 *3.8 ± 0.1 ***29.6 ± 0.9 ***36.1 ± 2.3 *SMM : Skeletal muscle mass, SMF : skeletal muscle functioning, 25(OH)D : 25-hydroxy vitamin D, Intact PTH : intact parathyroid hormone* : p < 0.05, ** : p < 0.01, *** : p < 0.001

Table 2. Effects of vitamin D-fortified milk intake between the sexes
Male
BaselineMean
Age (years)nSE

Female6 months
Mean

Baseline

SE

Mean

SE

46.4 ± 3.9

57.2 ± 4.4

16

18

6 months
Mean

SE

SMM
SMM (kg)

29.8 ± 2.3

29.8 ± 2.5

16.96 ± 1.6

19.6 ± 2.7

SMM (% of body weight)

44.1 ± 3.6

43.7 ± 4.7

30.7 ± 3.0

34.2 ± 4.1

SMM (kg / m2)

11.3 ± 0.9

10.8 ± 1.1

6.9 ± 0.6

7.7 ± 0.9

Creatinine height index (%)

88.7 ± 6.9

87.7 ± 9.2

78.3 ± 7.6

85.8 ± 10.6

9.6 ± 2.8

10.3 ± 3.5

7.1 ± 1.3

6.6 ± 1.3

SMF
Timed up and go test (sec)Walking speed (m / sec)

4.4 ± 0.7

4.3 ± 0.8

Handgrip strength (kg)36.2 ± 4.7

39.4 ± 4.2

Barthel index

87.2 ± 6.6

86.6 ± 6.5

Nitrogen (%)

80.3 ± 4.2

56.5 ± 4.0

Sodium (%)

91.4 ± 7.1

80.9 ± 7.2

Calcium (%)

20.3 ± 3.1

Phosphorus (%)*

3.3 ± 0.5

3.1 ± 0.3

26.2 ± 2.3

25.5 ± 2.5

84.7 ± 7.4

84.4 ± 7.6

56.2 ± 5.2

44.2 ± 4.3

75.5 ± 7.0

81.2 ± 6.8

16.2 ± 2.5

14.0 ± 2.2

12.5 ± 1.9

61.6 ± 4.0

67.2 ± 6.0

43.9 ± 4.5

56.5 ± 6.2*

Calcium (mg / dL)

9.3 ± 0.1

9.4 ± 0.1

9.1 ± 0.1

9.3 ± 0.1**

Phosphorus (mg / dL)

3.5 ± 0.1

3.6 ± 0.1

3.5 ± 0.1

4.0 ± 0.1**

Predicted intestinal absorption rate**

Concentration in serum

25(OH)D (ng / mL)

14.6 ± 1.5

30.0 ± 1.2

Intact PTH (pg / mL)

40.1 ± 3.3

36.1 ± 4.2***

12.4 ± 0.8

29.2 ± 1.4

42.2 ± 4.6

36.1 ± 2.3

**

***

SMM : Skeletal muscle mass, SMF : skeletal muscle functioning, 25(OH)D : 25-hydroxy vitamin D, Intact PTH : intact parathyroid hormone* : p < 0.05, ** : p < 0.01, *** : p < 0.001

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Y. Matsuura, et al. Vitamin D supplementation and skeletal muscle

primarily due to the loss of muscle mass (23-25). Muscle function decreases through the progression of sarcopenia, linking
vitamin D and different symptoms (26-29) because vitamin D
receptor expression decreases with age (30, 31). In our previous
study, an age-related decrease in skeletal muscle mass and its
cut-off levels for walking difficulty were clarified using 24-hourcreatinine excretion as a measure of total-body skeletal muscle
mass in a Japanese population including nursing home residents (4, 5). The skeletal muscle function, skeletal muscle mass(kg, kg / m2 , %BW) and CHI were also closely related to serum
25(OH)D levels (6).
In this study, the mean serum 25(OH)D concentration was low
in the study population at baseline and it markedly improved
after daily 1000-IU vitamin D-fortified milk intake for 6 months
in the 21-50 years, 51-75 years and total groups in both males
and females. The mean value (29.6 ng / mL) exceeded the recommendation for older adults of > 50 nmol / L (20 ng / mL) (32), but
47% of the participants achieved VDS, whereas 47% and 6% had
VDI and VDD, respectively. Vitamin D-fortified milk intake also
improved handgrip strength and physical performance in the
21-50 years group, but not in older groups. Vitamin D treatment
also did not improve muscle mass or nitrogen or calcium absorption rates. Regarding the relevance of vitamin D for muscle (33),
an improvement in the plasma 25(OH)D concentration may also
have played a role in the observed increase in muscle mass.
Japanese mean plasma 25(OH)D levels were 22.2 ng / mL
(55.6 ± 14.6 nmol / L) in 600 home-dwelling postmenopausal
women (34), 21 ng / mL (54.2 ± 29.0 nmol / L winter, 53.3 ± 30.3
nmol / L summer) in non-institutionalized elderly subjects requiring care (35) and 12 ng / mL (29.9 ± 13.1 nmol / L) in 133 physically inactive elderly subjects (84.6 ± 8.2 years old) living in nursing
homes (36). The average dietary intake of vitamin D was around
300 IU / d, which is approximately 150% and 88% of the adequate
intake by the Japan Dietary Reference Intake 2005 and 2020,
respectively (37). Supplementation with 5 μg (200 IU) and 20
μg (800 IU) of vitamin D daily for 30 days increased the plasma
25(OH)D level from 11.1 ± 3.2 ng / mL to 14.7 ± 3.6 ng / mL in 33
institutionalized subjects (87.3 ± 7.3 years old) (37) and 9.7 ±2.8 ng / mL to 19.3 ± 4.1 ng / mL in 32 physically disabled elderly(83.8 ± 7.6 years old) subjects living in nursing homes (38). Chelet al. (39) assessed the effects of daily supplementation with 600
IU of vitamin D for 4 months in elderly nursing home residents(84.3 ± 6.3 years old). The serum 25(OH)D level increased from
9.2 ng / mL to 28 ng / mL, and the percentage of subjects with aserum 25(OH)D level below 20 ng / mL was only 10.9%. Furthermore, Chapuy et al. (40) reported that daily supplementation with
800 IU of vitamin D in combination with l,200 mg of calcium increased the serum 25(OH)D level from 9.2 ng / mL to above 30.0
ng / mL after 6 months in older subjects (85.2 ± 7.1 years old).
These reports suggest that the daily administration of 800 IU ofvitamin D for several months is required for the correction of alow vitamin D status in the institutionalized elderly.
Furthermore, vitamin D-fortified milk intake signiﬁcantly
improved the handgrip strength and TUG, particularly inthe 21-50 years group. Of note, several cross-sectional studies
involving adults and younger humans demonstrated that sufficient vitamin D levels positively affected muscle strength (41-43).
Similarly, a positive association between sufficient vitamin D
status and higher handgrip muscle strength was reported ina study involving adolescent girls, revealing that girls with asufficient vitamin D status have a significantly higher handgrip
muscle strength than those with a poor vitamin D status (41).
Meta-analysis of randomized controlled studies (RTCs) using
vitamin D revealed vitamin D supplementation to be effective
for the global muscle strength, which was more marked in thosewith a 25(OH)D level < 30 nmol / l and those > 65 years of age(44).
Vitamin D supplementation at 1000 IU per day for one year
improved the hip muscle strength and TUG test in older womenwith VDI (45), increased the mean type II muscle ﬁber diameterand percentage of type II ﬁbers in elderly patients with poststroke hemiplegia (46), and increased the intra myonuclear
vitamin D receptor (VDR) concentration by 30% and total (type
I and II) muscle ﬁber size by 10% in mobility-limited elderly
women (47). On the other hand, increased 25(OH)D levels in the
51-75 years and 76 year or older groups were not related to theimprovement of muscle function in this study. The lack of effectmay be explained by our participants being vitamin D resistantand thus unable to improve their muscle strength. However, thepooled data from meta-analysis of RTCs suggested that vitamin
D supplementation increases upper and lower limb strength in
the healthy adult population ; therefore, vitamin D may have asignificantly positive effect on overall muscle function in humans(48).
Aging is associated with reduced serum vitamin D metabolites (49, 50), intestinal calcium absorption (51, 52) and age-related intestinal resistance to 1α,25(OH)2D3 (53-55). Regardlessof vitamin D supplementation, the intestinal calcium absorption
rate in this study decreased from 17.0 ± 1.9% to 14.3 ± 1.6%
and the calcium absorption rate increased from 78 mg / day atbaseline to 98 mg / day. Intestinal fractional calcium absorption(FCA) was previously assessed using a double isotope methodin post-menopausal osteoporosis patients (74.2 ± 2.9 years old)(56). The baseline FCA value of the participants was 21.5 ± 7.9%
and was significantly correlated with the serum 1,25(OH)2D
concentration, but not with the serum 25(OH)D concentration.
After treatment for 4 weeks, the FCA significantly increased by
59.5% in the eldecalcitol (ELD) (0.75 μg / day) group and by 45.9%(27.9 to 63.8%) in the 1α hydroxyl calcidiol (ALF) (1 μg / day)group, whereas no significant change in the plain vitamin D3(800 IU / day) group was noted. Therefore, the stimulation of FCA
by plain vitamin D3 requires a dose greater than 800 IU. Plainvitamin D3 administration was also not effective or induced only
small increases in FCA in a high-dose group (57, 58).
The age-related loss of skeletal muscle mass and function ispartly the result of impaired activation of postprandial muscle
protein synthesis. A post hoc study (59) demonstrated thatparticipants with higher baseline 25(OH)D concentrations (> 50
nmol / L) and dietary protein intake (> 1.0 g / kg / day) had greater
gains in appendicular muscle mass, skeletal muscle index andrelative appendicular muscle mass in response to the nutritional intervention. Protein synthesis is tightly controlled by the
Akt / mTOR pathway, which is activated by anabolic factors suchas insulin and amino acids (60). Vitamin D may interfere with
the insulin signaling pathway and insulin sensitivity (61, 62),
and promote skeletal muscle protein anabolism (61). Vitamin
D- and leucine-enriched nutrition may aid in the prevention andtreatment of muscle mass and strength loss in vivo because ofsynergistic effects of leucine and vitamin D on protein synthesis
in C2C12 myotubes (61). Therefore, synergy between vitamin D
and milk protein may help to prevent or counteract the loss ofmuscle mass and strength during aging. The improved handgrip
force and TUG test may be explained by the increased calcium
absorption and anabolic utility of nitrogen by vitamin D administration in this study.
This study has several limitations. The controlled before–after
design of this study did not include a control group. The lack ofrandomization is also a weak point related to the effects of vitamin D-fortified milk intake. The small sample size may prevent
us from establishing relevance. However, a minimum level of 50
nmol / L (20 ng / mL) of 25(OH)D must be reached in the generalelderly population and 75 nmol / L (30 ng / mL) is considered theThe Journal of Medical Investigation Vol. 68 August 2021
target in fragile study participants who have a higher risk offalls and fractures (63, 64). Therefore, daily 1,000-IU vitamin
D-fortified milk intake is a practical method to maintain aserum 25(OH)D level of 30 ng / mL, and to improve muscle
strength and physical function in the Japanese population.

CONFLICTS OF INTEREST AND ACKNOWLEDGEMENT
This study was financially supported by the Food Science
Institute Foundation (Ryoushoku- kenkyukai) and this is oneof the cooperating programs in the Kenshokai Group promotingsocial welfare. We express special thanks to all volunteers andnursing homes (Egao, Heart, Shoenburn) in the Kenshokai
Group for their kind support of this study.

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