Seven-month Wintertime Supplementation of 1200 IU Vitamin D Has No Effect on Hand Grip Strength in Young, Physically Active Males: A Randomized, Controlled Study.

doi:10.21203/rs.3.rs-660114/v1

Background

There has been a growing interest in the role of vitamin D for the well-being and physical performance of humans; however, there is a lack of long-term supplementation studies performed on members of the young, physically active, male population.

The hypothesis of the study was that vitamin D supplementation during wintertime will decrease the prevalence of critically low vitamin D blood serum levels and increase hand grip strength during the winter season among young male conscripts.

Study Design

Longitudinal, triple-blinded, randomized, placebo-controlled trial.

Methods

Fifty-three male conscripts from the Estonian Army were randomized into two groups: 27 to an intervention group and 26 to a placebo group. The groups were comparable in terms of their demographics. The intervention group received 1200 IU (30 µg) capsules of vitamin D3 and the control group received placebo oil capsules once per day. The length of the follow-up was seven months, from October 2016 until April 2017. Blood serum vitamin D (25(OH)D), parathyroid hormone (PTH), calcium (Ca), ionized calcium (Ca-i), testosterone and cortisol values and hand grip strength were measured four times during the study period.

Results

The mean 25(OH)D level decreased significantly in the control group to a critically low level during the study, with the lowest mean value of 22 nmol/l found in March 2017. At that time point, 65% in the control group vs 15% in the intervention group had 25(OH)D values of less than 25 nmol/l (p < 0.001). In the intervention group the levels of 25(OH)D did not change significantly during the study period. All other blood tests revealed no significant differences at any time point. The corresponding result was found for hand grip strength at all time points.

Conclusion

Long-term vitamin D supplementation during wintertime results in fewer conscripts in the Estonian Army with critically low serum vitamin D (25(OH)D) levels during the winter season. However, this did not influence their physical performance in the form of the hand grip strength test.

Trial registration

ClinicalTrials.gov Identifier: NCT04359524. Registered 20 April 2020 - Retrospectively registered. https://register.clinicaltrials.gov

Sports Medicine and Kinesiology

Nutrition & Dietetics

Military training

Hand grip

Vitamin D

Supplementation

Interest in the role of vitamin D in humans has been growing. Despite the large number of papers published during the last few decades, there is still a lack of knowledge in terms of seasonal variation and need for vitamin D supplementation in persons with a physically demanding lifestyle; this includes differences between age groups and gender subpopulations in both healthy persons and patients. The key role of vitamin D in musculoskeletal health is well known, especially the morbidity of its depletion [1-4]. However, it is also known that non-musculoskeletal disorders such as diabetes, metabolic disorders, infectious diseases caused by decreased immunity, autoimmune diseases, and hypertension are, potentially, adversely affected by a lack of vitamin D [5]. The effect of vitamin D on physical performance is still controversial and there is a lack of evidence in terms of its effect on hand grip strength [6-7].

Vitamin D is a unique form of hormone and has receptors in most cells and tissues. It has an enormously wide range of biological actions, including its key roles in calcium metabolism and bone modelling [8], and it has receptors in muscle tissue [9]. It is also known that it can affect the metabolism of testosterone and cortisol in the human body [10-12], both of which are known to be important in terms of physical performance and level of stress [13].

Uncovered skin contact with UV irradiation is the main mechanism of vitamin D synthesis in the human body [14]. Certain forms of vitamin D, such as D2 (found in mushrooms) and D3 (fish, eggs, meat), are the main natural food sources for humans [15]. Food supplements (capsules, tablets or food fortification) provide another option to cover the vitamin D needs of the human body. Vitamin D is inactive in the human body and needs a two-step hydroxylation: first in the liver to form 25(OH)D, and secondly in the kidneys to produce the biologically active form 1,25(OH)2D [16]. The main form of vitamin D in blood serum is 25(OH)D and this is used as an indicator of vitamin D status [17].

In a study by Funderburk et al. [18], subjects in a military environment were classified by their vitamin D status. In this study, vitamin D was considered sufficient if the serum 25(OH)D concentration was ≥ 75 nmol/l, insufficient if between 51 and 75 nmol/l, and deficient if < 50 nmol/l.

Based on an Estonian population study by Kull et al. in 2009 [19], critical deficiency was determined as a serum concentration of < 25 nmol/l. Furthermore, deficiency has been determined as < 50 nmol/l and insufficiency as < 75 nmol/l by the Endocrine Society (ES) [1]. There is no consensus concerning the normal level of serum 25(OH)D. Some studies have even defined the insufficiency boundary as high as 80 nmol/l [20,21].

Personal characteristics, such as skin pigmentation, age, type of clothing, use of sunscreen, physical and outdoor activity, and sun exposure, can prevent or promote vitamin D synthesis and will influence serum 25(OH)D levels [22].

A low level of vitamin D is a well-known problem in northern European countries. However, in recent studies, an increasing tendency towards this has also been reported in the southern part of Europe, and at higher northern latitudes (above 40°N; e.g. Madrid). Sun exposure during winter activates the dermal generation of vitamin D only at low levels or not at all; thus, the demand for dietary supplementation is increased [23,24].

A population-based study performed in Estonia found the mean serum 25(OH)D concentration to be 43.7 nmol/l during winter and 59.3 nmol/l during summer. The authors of that study stated that vitamin D deficiency is highly prevalent throughout the year in this population, who live at a latitude of 59°N without vitamin D dairy product supplementation [19]. A large Finnish-population study also found vitamin D deficiency in all age groups during the winter season [25] and there are similar seasonal variations reported in other European studies [1,2,5,24-29].

Routine dietary fortification of vitamin D is common practice in Norway, Sweden and Finland. These countries therefore have better basic levels of serum 25(OH)D among their populations than Estonia [25,28-30]. There is no official dairy fortification with vitamin D in Estonia, which increases the risk of having a low level of serum 25(OH)D [19].

Muscle fitness is crucial in military service. To measure physical fitness, the hand grip strength test is easy. It is a widely used test to measure muscle fitness in all age groups [31-36] and is often used as an indicator of general health. Carswell et al. [4] found evidence that low 25(OH)D levels were negatively related to muscle fitness in the UK army. Other studies show that lower muscle fitness and hand grip strength are related to higher risk of cardiovascular disease events and mortality in middle-aged individuals [37] and an increased suicide risk in adolescents [38]. There is evidence that vitamin D supplementation has a positive effect on upper limb muscle strength [6]. There is lack of knowledge on how vitamin D deficiency and supplementation affects hand grip strength in young, healthy individuals, such as conscripts.

Therefore, a prospective, randomized, blinded study in young male conscripts in the Estonian army was designed. The hypothesis of the study was that without oral vitamin D supplementation, vitamin D deficiency occurs during the winter season and that this can cause a decrease in hand grip strength.

The primary outcome of the study was the serum levels of 25(OH)D during the study period. The secondary outcome of the study was the hand grip strength.

All conscripts (n = 403) entering military service in October 2016 at the Kuperjanov Battalion, Võru, Estonia (situated at a latitude of 58°N, which corresponds to that of southern Alaska in the US and Quebec in Canada) were asked to participate in the first briefing of this study. The recruits were informed of the purpose of the study, recruitment criteria and follow-up methods. Only those who volunteered to participate in the study and signed informed consent after this first briefing were included in the study. A total of 65 conscripts volunteered to participate initially and 63 of these returned their informed consent.

Ten conscripts¸ five in the intervention group and five in the control group; were later excluded from the study due to premature cessation of their military service: two for mental health problems, two for lower-back pain, one for polyarthritis and five for other medical reasons. Data from 53 conscripts, all of Caucasian origin, were included in the final analysis: 27 in the intervention group and 26 in the control group. The demographics of the study groups are presented in Table 1.

The only exclusion criterion was the inability to continue military service, for any reason, during the seven-month follow-up period.

A flow chart of the study is presented in Figure 1.

The study was approved by the Research Ethics Committee of the University of Tartu no. 262/T-28 and 264M-14 and funded by grant no. R-002 of Estonian Defence Forces.

Table 1. Demographics at the baseline and during follow-up of study groups.

Intervention group

Placebo group

Significance

Number of participants

27

26

Age (years)
Median (range)

Mean (SD)

21 (19–27)

20.8 (1.7)

21 (19–26)

21.2 (2.0)

n.s. (0.38)

Length (cm)
Median (range)

Mean (SD)

180 (167–191)

180 (6.9)

181 (163–191)

179.0 (7.7)

n.s. (0.49)

Weight baseline (October 2016) (kg)

Median (range)

Mean (SD)

72.0 (55.7–97.7)

74.0 (10.8)

75.6 (49.3–95.2)

74.5 (11.1)

n.s. (0.89)

BMI baseline (October 2016) kg/m²

Median (Range)

Mean (SD)

22.0 (19.5–28.2)

22.7 (2.4)

23.1 (18.1–26.1)

23.2 (2.6)

n.s. (0.48)

Weight follow-up III (April 2017) (kg)

Median (range)

Mean (SD)

75.0 (61.4–100.2)

76.8 (10.3)

77.2 (56.9–96.0)

77.4 (10.6)

n.s. (0.85)

BMI follow-up III (April 2017) kg/m²

Median (Range)

Mean (SD)

23.3 (19.6–28.9)

23.5 (2.1)

24.1 (20.4–28.1)

24.1 (2.4)

n.s. (0.37)

Abbreviations: SD, standard deviation; BMI, body mass index, n.s., non-significant

Study design and data collection

A longitudinal, triple-blinded, randomized, placebo-controlled trial (ClinicalTrials.gov NCT04359524) with a seven-month follow-up period from October 2016 until April 2017 was performed.

Body mass (kg) and height (cm) of the conscripts were measured two times by the same nurse at the Kuperjanov Battalion medical center using standardized equipment, and their body mass index (BMI) was calculated in kg/m².

Computed randomization was used to divide conscripts into two groups: either the intervention group, in which conscripts received vitamin D3 capsules (1200 IU/30 µg), or the control group, in which conscripts received a placebo (oil capsules). Both types of capsules were administered once per day, in the morning before breakfast, for seven months. Standardized coded packages (three per conscript) and capsules (100 per package) were manufactured on special order by Innopharma A/S (Denmark). No commercial sponsoring was involved. The key to the package code numbers was stored in a computer database until the unblinding of the participants.

A dosage of 1200 IU of vitamin D3 per day was used, because this was the maximum recommended daily supplementation dosage stated by the Estonian State Agency of Medicines (Personal communication. Based on regulation No. 59 of the Minister of Social Affairs of the Republic of Estonia, April 13^th, 2005) at the time of the study.

The hand grip test was performed using a validated hydraulic hand dynamometer (Lafayette Instrument Co., USA). Measurements were taken from each participant in the standing position, arms at the side, not touching the body, with the elbow slightly bent. The participant squeezed the dynamometer with as much force as possible. The best result of three trials, with pause of about 10–20 seconds between the trials, was recorded in kilograms. The same procedure was performed for both hands.

Blood serum values of vitamin D (25(OH)D), parathyroid hormone (PTH, normal 1.48–7.83 pmol/l), calcium (Ca, normal 2.15–2.6 mmol/l), ionized calcium (Ca-i, normal 1.12–1.32 mmol/l), testosterone (normal 8.4–28.7 nmol/l) and cortisol (normal 138–690 nmol/l) were measured four times during the study period: first in October 2016 to provide baseline values, then subsequently in December 2016, March 2017 and April 2017. All blood samples were overnight fasting tests, collected on the same day of the week, and all within the same hour, under standardized conditions.

Laboratory measurements

Serum samples for clinical chemistry analysis were collected in serum clot activator tubes (BD Vacutainer SST II Advance Plus Blood Collection Tubes, Becton Dickinson and Company, New Jersey, United States). Calcium measurements were performed using the spectrophotometry method (ADVIA® 1800 Clinical Chemistry System, Siemens Healthcare GmbH, Erlangen, Germany). Ionized calcium measurements were performed using ion selective electrodes (AVL 9180 Electrolyte Analyzer, Roche Diagnostics, Germany). The direct chemiluminescent immunoassay method was used for measurement of PTH (ADVIA Centaur XP, Siemens Healthcare GmbH, Erlangen, Germany). Measurements of 25(OH)D were performed using the direct chemiluminescent immunoassay method (LIAISON XL, DiaSorin S.p.A, Saluggia VC, Italy). The direct chemiluminescent immunoassay method was used for measurement of testosterone (ADVIA Centaur TSTII assay, Siemens Healthcare GmbH, Erlangen, Germany). The solid-phase competitive chemiluminescent enzyme immunoassay method was used for measurement of cortisol (IMMULITE 2000 Cortisol, Siemens Healthcare Diagnostics Products Ltd. United Kingdom). All analyses were performed by Synlab Estonia.

Power calculation

The primary variable of the study was the level of 25(OH)D in the serum. In the absence of available pilot data, a pragmatic decision was taken in the power analysis; a difference of 20 nmol/l (i.e. less than the increments of 25 nmol/l in the study by Funderburk et al. (18) between the intervention and the control group was considered to be the meaningful detectable difference. For example, if the SD would be 25 nmol/l, then 26 participants would be needed in each group to reach a power of 80%. Correspondingly if the difference in hand grip strength would be 6 kilograms between the study groups, and the SD 8 kilograms, then 29 participants would be needed in each group to reach a power of 80%. Initially, 65 participants were recruited to the study to allow for dropouts.

Statistical analysis

Statistical analysis was performed using SPSS version 25.

The blood serum values and the hand grip strength in the study groups were described by medians, ranges, means and standard deviations (SD). Differences in mean values of the variables of interest between the groups were evaluated using an unpaired t-test. The within-group comparisons over time were performed using a paired t-test. Distributions of categorical variables were described by absolute numbers and percentages and compared between groups using the Fisher exact test. Statistical significance was set at p < 0.05.

The mean baseline values of 25(OH)D in October 2016 were 50.0 nmol/l in the control group and 48.3 nmol/l in the intervention group (p = 0.55). At all subsequent time points, the control group had a significantly lower value of 25(OH)D (p < 0.001).

A significant decrease in the levels of 25(OH)D compared with the baseline were found at all time points in the control group, with a lowest average level of 22 nmol/l in March 2017 (p < 0.001). In the intervention group, no significant decrease over time was seen. The average seasonal values of 25(OH)D for both groups are presented in Table 2.

In April 2017, 11/26 (42%) in the control group vs 1/27 (4%) in the intervention group had 25(OH)D values < 25 nmol/l (p < 0.001). There were no conscripts with values higher than 50 nmol/l in the control group in April 2017 (Tables 3 and 4). Seasonal within-group variation in 25(OH)D levels is presented in Figure 2.

No significant differences in Ca, Ca-i, PTH, testosterone and cortisol levels were revealed between the study groups at any time point (Table 2). One participant in the control group had abnormally high PTH values throughout the study period, with serum 25(OH)D values lower than 25 nmol/l at all time points. No significant differences at any time points were revealed in the hand grip strength tests of either hand between the study groups (Table 5).

Table 2. Vitamin D 25(OH)D, PTH, testosterone, cortisol, calcium and ionized calcium serum level results.

Blood serum tests (normal values)		Baseline (October 2016)		Follow-up I (December 2016)		Follow-up II (March 2017)		Follow-up III (April 2017)
		Intervention group	Placebo group	Intervention group	Placebo group	Intervention group	Placebo group	Intervention group	Placebo group
Vitamin D 25(OH)D (75 > nmol/l) Median (range) Mean (SD)		47.8 (19.3–93.9) 48.3 (18.0)	50.0 (22.3–88.7) 49.8 (20.0)	62.5 (19.1–91.0) 59.8 (15.9)	36.8 (15.9–66.9) 36.4 (14.2)	53.0 (12.1–88.4) 50.2 (19.8)	20.2 (11.1–37.3) 22.0 (7.7)	52.2 (18.6–89.1) 53.6 (18.3)	28.9 (16.9–49.9) 29.9 (9.3)
Parathyroid hormon (1.48–7.83 pmol/l) Median (range) Mean (SD)		4.5 (1.7–10.1) 4.8 (2.1)	4.4 (0.9-9.9) 4.5 (2.4)	3.3 (0.8–7.4) 3.6 (1.7)	3.9 (0.7–12.9) 4.3 (2.4)	4.3 (1.2–6.0) 4.0 (1.6)	4.3 (1.8–20.1) 5.0 (3.6)	4.5 (1.8–8.2) 4.7 (1.8)	4.3 (2.2–8.8) 4.9 (2.1)
Testosterone (8.4–28.7 nmol/l) Median (range) Mean (SD)		15.4 (10.8–29.6) 17.4 (5.3)	16.2 (6.1–31.0) 17.4 (5.7)	20.0 (5.2–26.6) 18.7 (5.3)	18.9 (11.7–30.0) 19.3 (4.0)	19.1 (8.3–26.0) 18.5 (4.2)	21.0 (5.7–28.4) 20.5 (4.8)	20.5 (10.1–32.5) 21.3 (5.9)	20.1 (13.0–28.4) 20.3 (4.1)
Cortisol (138–690 nmol/l) Median (range) Mean (SD)		527.0 (400–615) 517.3 (62.7)	506.5 (348–670) 513.7 (69.8)	538 (450–756) 554.0 (70.1)	561.5 (292–676) 536.1 (70.1)	452.0 (251–629) 457.7 (105.1)	452.5 (114–577) 438.6 (94.4)	488.0 (265–668) 482.4 (85.8)	473.5 (287–599) 470.3 (76.6)
Calcium (2.15–2.6 mmol/l) Median (range) Mean (SD) degrees		2.37 (2.28–2.50) 2.39 (0.07)	2.38 (2.23–2.53) 2.37 (0.07)	2.28 (2.18–2.36) 2.28 (0.05)	2.25 (2.13–2.43) 2.26 (0.06)	2.36 (2.29–2.48) 2.36 (0.05)	2.36 (2.24–2.46) 2.35 (0.06)	2.29 (2.11–2.42) 2.28 (0.07)	2.26 (2.12–2.34) 2.24 (0.05)
Calcium ionized (1.12–1.32 mmol/l) Median (range) degrees Mean (SD) degrees		1.23 (1.15–1.31) 1.23 (0.04)	1.22 (1.15–1.28) 1.21 (0.03)	1.25 (1.20–1.32) 1.25 (0.03)	1.24 (1.17–1.32) 1.25 (0.04)	1.23 (1.08–1.33) 1.22 (0.04)	1.23 (1.18–1.28) 1.23 (0.03)	1.23 (1.18–1.29) 1.24 (0.03)	1.23 (1.15–1.29 1.23 (0.03)
Significance between groups:
Vitamin D 25(OH)D	n.s (0.55)			< 0.001		< 0.001		< 0.001
Parathyroid hormone	n.s. (0.60)			n.s. (0.20)		n.s. (0.14)		n.s. (0.75)
Testosterone	n.s. (1.0)			n.s. (0.65)		n.s. (0.12)		n.s. (0.46)
Cortisol	n.s. (0.85)			n.s. (0.43)		n.s. (0.5)		n.s. (0.59)
Calcium	n.s. (0.30)			n.s. (0.22)		n.s. (0.61)		0.05
Ionized calcium	n.s. (0.23)			n.s. (0.58)		n.s. (0.58)		n.s. (0.37)

Abbreviations: SD, standard deviation, n.s, non-significant

Table 3. Vitamin D 25(OH)D serum levels divided into subgroups according to Funderbunk et al. [18]

	Baseline (October 2016)		Follow-up I (December 2016)		Follow-up II (March 2017)		Follow-up III (April 2017)
	Intervention group (n = 27)	Placebo group n = (26)	Intervention group (n = 27)	Placebo group (n = 26)	Intervention group (n = 27)	Placebo group (n = 26)	Intervention group (n = 27)	Placebo group (n = 26)
0–24 nmol/l (%)	3 (11.1)	3 (11.5)	1 (3.7)	8 (30.8)	4 (14.8)	17 (65.4)	1 (3.7)	11 (42.3)
25–49 nmol/l (%)	13 (48.1)	10 (38.5)	8 (29.6)	14 (53.8)	8 (29.6)	9 (34.6)	11 (40.7)	15 (57.7)
50–74 nmol/l (%)	9 (33.3)	10 (38.5)	14 (51.9)	4 (15.4)	12 (44.4)	-	11 (40.7)	-
> 75 nmol/l (%)	2 (7.4)	3 (11.5)	4 (14.8)	-	3 (11.1)	-	4 (14.8)	-
	Significance between groups:
	n.s. (0.84)		p = 0.015		p < 0.001		p < 0.001

Table 4. Vitamin D 25(OH)D serum level comparison over time within groups.

	Baseline (October 2016) vs Follow-up I (December 2016)	Baseline (October 2016) vs Follow-up II (March 2017)	Baseline (October 2016) vs Follow-up III (April 2017)
Intervention group	0.012	n.s (0.32)	n.s. (0.12)
Placebo group	< 0.001	< 0.001	< 0.001

Abbreviations: n.s, non-significant

Table 5. Hand grip strength results for both hands during the study period.

	Baseline (October 2016)		Follow-up I (December 2016)		Follow-up II (March 2017)		Follow-up III (April 2017)
	Intervention group	Placebo group	Intervention group	Placebo group	Intervention group	Placebo group	Intervention group	Placebo group
Handgrip right Median (range) Mean (SD)	48.0 (29–72) 48.0 (9.8)	50 (28–72) 49.1 (10.1)	50.0 (30–65) 50.1 (7.6)	50.0 (30–70) 50.9 (8.5)	52.0 (30–80) 52.6 (9.6)	53.0 (30–70) 52.5 (10.0)	51.0 (28–84) 51.0 (9.0)	50.5 (34–68) 51.8 (9.0)
Handgrip left Median (range) Mean (SD)	46.0 (32–66) 45.9 (8.3)	50.0 (29–68) 47.8 (9.3)	48.0 (32–60) 47.9 (6.7)	49.0 (32–70) 49.5 (9.3)	50 (38–68) 50.8 (7.3)	52.0 (32–70) 50.8 (8.7)	49.0 (38–75) 49.9 (9.2)	50.0 (32–66) 50.7 (8.9)
Handgrip right	n.s. (0.70)		n.s. (0.74)		n.s. (0.97)		n.s. (0.78)
Handgrip left	n.s. (0.45)		n.s. (0.48)		n.s. (1.0)		n.s. (0.76)

Abbreviations: SD, standard deviation. n.s non-significant

The main finding of the present study is that seven-month vitamin D3 supplementation results in fewer conscripts in the Estonian Army with critically low serum vitamin D (25(OH)D) levels during the winter season; however, this supplementation did not appear to affect the hand grip strength at any time point during the study period.

Moreover, the decrease in the levels of 25(OH)D in the placebo group of this study reveals how crucial vitamin D supplementation is at Nordic latitudes, because the majority (65%) of the subjects in the placebo group presented critically low levels in March 2017, and none of them reached to 50 nmol/l in April.

In 2016, the European Food Safety Authority (EFSA) panel considered that a serum 25(OH)D concentration of 50 nmol/l is a suitable target value for all population groups; this should be possible to achieve with a dietary intake of 15 µg/day (equal to 600 IU) [39].

The present study shows that during the winter season the regular diet administered by the Estonian Army to conscripts does not provide enough vitamin D to achieve the 50 nmol/l level suggested by the EFSA panel. The exact amount of vitamin D in the regular Estonian Army diet is not known. However, the diet of the conscripts is based on guidelines from professional dieticians who have calculated the daily requirements of energy, minerals and vitamins (Personal communication. "Regulation of dietary norms for soldiers”, regulation no. 240 of the Commander of the Defense Forces of the Estonian Army, September 13^th, 2013).

A similar finding of widespread vitamin D deficiency among soldiers was presented in a recent prospective cohort study performed in the US Army [40]. Another recent prospective cohort study in the UK Army reported that vitamin D is clearly associated with endurance performance; however, it showed that power and strength are not affected by vitamin D [4].

Vitamin D has a stimulating effect on muscle protein synthesis [9] and has been shown to have a crucial biomolecular role in skeletal muscle activation and, thus, muscle function [3,41]. Therefore, vitamin D deficiency may have a detrimental effect during physically demanding situations, such as military service.

Testosterone and cortisol have crucial roles in muscle fitness, physical performance and general health [13]. Vitamin D is linked to testosterone production in the human body and supplementation of vitamin D has shown a positive effect on testosterone levels [42]. It is also known that increased levels of testosterone can improve physical performance [43]. Cortisol, commonly called the stress hormone, is one of the glucocorticoids, and it has an important role in regulating muscle function, energy homeostasis, metabolism, and adaptation for physical exercises [44]. However, increased levels of cortisol can decrease physical performance ability [45]. Interestingly, no significant differences between the study groups were revealed in either testosterone or cortisol levels at any time point in the present study. This might be due to the fact that either the study group was small or that summertime monitoring was not performed. Lombardi et al. [46] found higher levels of cortisol during wintertime and higher levels of testosterone during summertime in a group of 167 elite football players, which also correlated with vitamin D seasonal differences. Mielgo-Ayuso et al. [43] found that short-term (eight weeks) supplementation with 3000 IU vitamin D in 36 elite male rowers did not increase muscle recovery; however, they did find that serum 25(OH)D levels were predictors of both anabolic and catabolic hormone levels. On the other hand, a recent study of 50 young male ice hockey players found no statistically significant associations between 25(OH)D and testosterone and cortisol concentrations in a single blood test in the month of October [47]. In line with this, Worzosek et al. [48], in a study of 55 male athletes who received 12 weeks of 2000 IU vitamin D supplementation, found no effect on the levels of testosterone, estradiol or cortisol, which is similar to the findings of the present study.

Hand grip strength results in the present study are similar to other European and North American population-based studies [49]. Despite low 25(OH)D levels in the springtime in the control group, there were no differences in the hand grip strength of either hand between the study groups.

There is still lack of knowledge in terms of the role of vitamin D for upper limb muscle strength in the general population. The studies of Haslam et al. [50] and Wang et al. [51] showed that vitamin D deficiency is related to loss of hand grip strength in the older population and there is evidence that vitamin D also has a key role in younger age groups for upper limb muscle strength [6,52-54].

For people involved in physically demanding activities, such as army service, there is probably a higher demand for vitamin D supplementation than shown in previous studies. This is supported by multiple studies showing vitamin D deficiency in army recruits [26,27,40,55]. Willis et al. [56] found that a surprisingly high proportion of athletes have a vitamin D deficiency; 77% of German gymnasts had 25(OH)D levels below 35 ng/ml, and 37% had a critical deficiency value of < 10 ng/ml [56].

In a study by Allison et al. [57], it was reported that severely vitamin-D-deficient athletes have smaller hearts than those athletes with moderate vitamin D deficiency or insufficiency, but a similar effect was not observed in healthy, non-athletic controls.

Hughes et al. [58] showed in a study in the US Army, that under military training, bone resorption is increased and bone formation slowed down. In their study, bone metabolism was normalized two to six weeks after cessation of training [58]. According to this prospective study of 3787 soldiers participating in military operations over 15 months, 19% needed an orthopedic consultation, and for 4% orthopedic surgery was indicated [59]. Vitamin D has a very important role in bone metabolism, and it has been shown that vitamin D and calcium supplementation can prevent stress fractures during military service. A large controlled, randomized, double-blind study of 5201 female navy recruits in the US Army demonstrated that calcium and vitamin D supplementation resulted in a 20% lower incidence of stress fractures than in a control group [60].

Vitamin D deficiency also has a crucial role in the human immune system at the cellular level [61]. Laaksi et al. [27] detected an association between low 25(OH)D levels and occurrence of respiratory infections in 800 Finnish conscripts. In a randomized, double-blinded study, performed by the same authors on 164 conscripts over a six-month period during the winter season, a 400 IU vitamin D supplementation was compared to a placebo. This study revealed that the frequency of acute respiratory infections was significantly lower in the supplementation group [25]. Similar findings were detected in college athletes in the USA in a prospective follow-up study monitoring vitamin D status [4].

The recommendations of the ES [1] are similar to the EFSA 2016 recommendations [39]for young, healthy males under normal civil conditions. However, the daily Recommended Dietary Allowance (RDA) of the ES for risk groups is 1500–2000 IU [39]. Military service can be considered a risk factor for vitamin D deficiency [9,57,58]. Despite the fact that a double dose based on ES or EFSA recommendations was used in the present study, it was still insufficient to improve the 25(OH)D serum levels in Estonian Army conscripts during an intensive winter-season training period. It has been debated that under physically demanding situations such as athletic performance, the usual amounts of vitamin D supplementation may be insufficient. It has been speculated that the supplementation amounts should instead be 1000–2000 IU of vitamin D per day [56].

In a cross-sectional study based on all age groups of the Estonian population, Kull et al. [19] found low values of 25(OH)D, similar to those in the control group of the present study.

The northern latitude of Estonia, as well as the high physical demand of military service, apparently exposes conscripts to an increased risk of vitamin D deficiency. Thus, higher doses may be needed in order to prevent vitamin D deficiency in this group.

It would be interesting to follow young individuals in the northern latitudes over a longer period of time to evaluate the effect of vitamin D supplementation for their performance and well-being.

Future studies should not only be done on conscripts, but on the general population as well. Furthermore, higher vitamin D supplementation doses could be used for this, since up to 4000 IU per day is now authorized by the Estonian State Agency of Medicines.

In terms of PTH levels, during the present study there were no significant changes detected over time. This is in concordance with earlier reports that PTH decreases during the summer–autumn season, [62] which was unfortunately outside the follow-up period of the present study.

It is known that excessively high doses of Vitamin D or blood serum levels of 25(OH)D together with low calcium intake can increase the calcium level outside the normal range, causing increase in bone resorption and decrease in bone mineralization [63]. There is also a risk of hypercalcemia causing general gastrointestinal problems such as constipation and hypercalciuria with renal calculi. Myocardial infarction, stroke, vascular disease and even death have been reported after intake of calcium supplements [64]. In the present study, the calcium levels were normal during the study period, indicating that a dose of at least 1200 IU can safely be used.

The strengths of this study lie in its randomized and blinded design, the homogenous test group, and the relatively long follow-up period over the winter season with four physical performance test occasions. Until now, there have been only short-term (8–12 weeks of follow-up), randomized studies performed on soldiers under physically demanding activities to examine the effect of vitamin D supplementation [4,40,55,65]. The present study examines the effect of supplementation during the whole winter season.

The study population was well standardized in terms of conditions, such as the season of the year, age, sex, daily food consumption, state of dress, and physical activity. This provided an ideal opportunity to study the effect of vitamin D supplementation on serum levels of 25(OH)D and physical performance in the form of the hand grip test.

The limitations of the study include the small groups, relatively short wintertime follow-up of 7 months, the use of only one supplementation dosage, only one physical performance test and the exclusion of female subjects. Due to participant dropouts, the study is underpowered for the secondary outcome variable, the hand grip test. Furthermore, many conscripts chose not to participate. However, it can be assumed that there are either no or only small differences between conscripts because of the homogeneity of soldiers in the Estonian Army. Lastly, the generalizability of the study is limited to military personal in relatively high northern or southern latitudes.

Long-term vitamin D3 supplementation results in fewer conscripts in the Estonian Army with critically low serum vitamin D (25(OH)D) levels during the winter season. However, this did not influence their physical performance in the form of the hand grip strength test.

ES Endocrine Society;

PTH parathyroid hormone;

Ca calcium;

Ca-i ionized calcium; (Ca-i),

SD standard deviation;

BMI body mass index;

n.s non-significant;

EFSA European Food Safety Authority;

RDA Recommended Dietary Allowance.

Ethics approval and consent to participate.

The study was approved by the Research Ethics Committee of the University of Tartu no. 262/T-28 and 264M-14.

Consent of publication.

“Not applicable”

Availability of data and materials.

The datasets generated and/or analyzed during the current study are available in the datadoi.ee University of Tartu Library repository, https://datadoi.ee/handle/33/342, http://dx.doi.org/10.23673/re-284

Competing interests.

Leho Rips – Lecturing for Sanofi Estonia

Alar Toom – No conflicts

Rein Kuik – No conflicts

Ahti Varblane – No conflicts

Hanno Mölder – No conflicts

Helena Gapeyeva – No conflicts

Marika Tammaru – No conflicts

Mart Kull – No conflicts

Vahur Ööpik – No conflicts

Jüri-Toomas Kartus – Lecturing for ConMed, Sweden

Madis Rahu – No conflicts

Funding.

This study was supported by the Estonian Defense Forces, funding agreement No 0.4-2.5/16/1321.

Authors` contributions.

Leho Rips – Designed research, analyzed data, writing of paper

Alar Toom – Analyzed data, writing of paper

Rein Kuik – Designed research

Ahti Varblane – Designed reserch, conducted research

Hanno Mölder – Conducted research

Helena Gapeyeva – Designed research, conducted research

Mart Kull – Analyzed data, writing of paper

Vahur Ööpik – Designed research

Jüri-Toomas Kartus – Primary responsibility for final content, writing of paper

Madis Rahu – Primary responsibility for final content, writing of paper

Acknowledgements.

The authors thank the conscripts who participated in the study as well as the command staff of the Kuperjanov Single Infantry Battalion for allowing access to the conscripts, sergeant major Terje Rammo for excellent technical assistance and Ninni Sernert for critical review and statistical analyses.

1. Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, et al. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2011 Jul;96(7):1911–30.

2. Sinha A, Hollingsworth KG, Ball S, Cheetham T. Improving the vitamin D status of vitamin D deficient adults is associated with improved mitochondrial oxidative function in skeletal muscle. J Clin Endocrinol Metab. 2013 Mar;98(3):E509-13.

3. Halliday TM, Peterson NJ, Thomas JJ, Kleppinger K, Hollis BW, Larson-Meyer DE. Vitamin D status relative to diet, lifestyle, injury, and illness in college athletes. Med Sci Sports Exerc. 2011 Feb;43(2):335–43.

4. Carswell AT, Oliver SJ, Wentz LM, Kashi DS, Roberts R, Tang JCY, et al. Influence of Vitamin D Supplementation by Sunlight or Oral D3 on Exercise Performance. Med Sci Sports Exerc. 2018 Dec;50(12):2555–64.

5. Płudowski P, Karczmarewicz E, Bayer M, Carter G, Chlebna-Sokół D, Czech-Kowalska J, et al. Practical guidelines for the supplementation of vitamin D and the treatment of deficits in Central Europe - recommended vitamin D intakes in the general population and groups at risk of vitamin D deficiency. Endokrynol Pol. 2013;64(4):319–27.

6. Stockton KA, Mengersen K, Paratz JD, Kandiah D, Bennell KL. Effect of vitamin D supplementation on muscle strength: a systematic review and meta-analysis. Osteoporos Int J Establ Result Coop Eur Found Osteoporos Natl Osteoporos Found USA. 2011 Mar;22(3):859–71.

7. Zhang L, Quan M, Cao Z-B. Effect of vitamin D supplementation on upper and lower limb muscle strength and muscle power in athletes: A meta-analysis. PloS One. 2019;14(4):e0215826.

8. Giustina A, Adler RA, Binkley N, Bollerslev J, Bouillon R, Dawson-Hughes B, et al. Consensus statement from 2nd International Conference on Controversies in Vitamin D. Rev Endocr Metab Disord. 2020 Mar;21(1):89–116.

9. Girgis CM, Clifton-Bligh RJ, Hamrick MW, Holick MF, Gunton JE. The roles of vitamin D in skeletal muscle: form, function, and metabolism. Endocr Rev. 2013 Feb;34(1):33–83.

10. Pilz S, Frisch S, Koertke H, Kuhn J, Dreier J, Obermayer-Pietsch B, et al. Effect of vitamin D supplementation on testosterone levels in men. Horm Metab Res Horm Stoffwechselforschung Horm Metab. 2011 Mar;43(3):223–5.

11. Kaji H. Interaction between Muscle and Bone. J Bone Metab. 2014 Feb;21(1):29–40.

12. de Angelis C, Galdiero M, Pivonello C, Garifalos F, Menafra D, Cariati F, et al. The role of vitamin D in male fertility: A focus on the testis. Rev Endocr Metab Disord. 2017 Sep;18(3):285–305.

13. Casto KV, Edwards DA. Testosterone, cortisol, and human competition. Horm Behav. 2016 Jun;82:21–37.

14. Holick MF, Tian XQ, Allen M. Evolutionary importance for the membrane enhancement of the production of vitamin D3 in the skin of poikilothermic animals. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3124–6.

15. Lamberg-Allardt C. Vitamin D in foods and as supplements. Prog Biophys Mol Biol. 2006 Sep;92(1):33–8.

16. DeLuca HF. Overview of general physiologic features and functions of vitamin D. Am J Clin Nutr. 2004 Dec;80(6 Suppl):1689S-96S.

17. Calvo MS, Whiting SJ, Barton CN. Vitamin D intake: a global perspective of current status. J Nutr. 2005 Feb;135(2):310–16.

18. Funderburk LK, Daigle K, Arsenault JE. Vitamin D status among overweight and obese soldiers. Mil Med. 2015 Feb;180(2):237–40.

19. Kull M, Kallikorm R, Tamm A, Lember M. Seasonal variance of 25-(OH) vitamin D in the general population of Estonia, a Northern European country. BMC Public Health. 2009 Jan 19;9:22.

20. Bischoff-Ferrari HA, Giovannucci E, Willett WC, Dietrich T, Dawson-Hughes B. Estimation of optimal serum concentrations of 25-hydroxyvitamin D for multiple health outcomes. Am J Clin Nutr. 2006 Jul;84(1):18–28.

21. Dawson-Hughes B, Heaney RP, Holick MF, Lips P, Meunier PJ, Vieth R. Estimates of optimal vitamin D status. Osteoporos Int J Establ Result Coop Eur Found Osteoporos Natl Osteoporos Found USA. 2005 Jul;16(7):713–6.

22. Cashman KD, Kiely M. Recommended dietary intakes for vitamin D: Where do they come from, what do they achieve and how can we meet them? J Hum Nutr Diet Off J Br Diet Assoc. 2014 Oct;27(5):434–42.

23. Holick MF. Vitamin D: A millenium perspective. J Cell Biochem. 2003 Feb 1;88(2):296–307.

24. Spiro A, Buttriss JL. Vitamin D: An overview of vitamin D status and intake in Europe. Nutr Bull. 2014 Dec;39(4):322–50.

25. Laaksi IT, Ruohola J-PS, Ylikomi TJ, Auvinen A, Haataja RI, Pihlajamäki HK, et al. Vitamin D fortification as public health policy: significant improvement in vitamin D status in young Finnish men. Eur J Clin Nutr. 2006 Aug;60(8):1035–8.

26. Laaksi I, Ruohola J-P, Mattila V, Auvinen A, Ylikomi T, Pihlajamäki H. Vitamin D supplementation for the prevention of acute respiratory tract infection: a randomized, double-blinded trial among young Finnish men. J Infect Dis. 2010 Sep 1;202(5):809–14.

27. Laaksi I, Ruohola J-P, Tuohimaa P, Auvinen A, Haataja R, Pihlajamäki H, et al. An association of serum vitamin D concentrations < 40 nmol/L with acute respiratory tract infection in young Finnish men. Am J Clin Nutr. 2007 Sep;86(3):714–7.

28. Lamberg-Allardt CJ, Outila TA, Kärkkainen MU, Rita HJ, Valsta LM. Vitamin D deficiency and bone health in healthy adults in Finland: could this be a concern in other parts of Europe? J Bone Miner Res Off J Am Soc Bone Miner Res. 2001 Nov;16(11):2066–73.

29. Lips P, Hosking D, Lippuner K, Norquist JM, Wehren L, Maalouf G, et al. The prevalence of vitamin D inadequacy amongst women with osteoporosis: an international epidemiological investigation. J Intern Med. 2006 Sep;260(3):245–54.

30. Lips P, Duong T, Oleksik A, Black D, Cummings S, Cox D, et al. A global study of vitamin D status and parathyroid function in postmenopausal women with osteoporosis: baseline data from the multiple outcomes of raloxifene evaluation clinical trial. J Clin Endocrinol Metab. 2001 Mar;86(3):1212–21.

31. Alahmari KA, Kakaraparthi VN, Reddy RS, Silvian PS, Ahmad I, Rengaramanujam K. Percentage difference of hand dimensions and their correlation with hand grip and pinch strength among schoolchildren in Saudi Arabia. Niger J Clin Pract. 2019 Oct;22(10):1356–64.

32. Burdukiewicz A, Pietraszewska J, Andrzejewska J, Chromik K, Stachoń A. Asymmetry of Musculature and Hand Grip Strength in Bodybuilders and Martial Artists. Int J Environ Res Public Health. 2020 Jun 30;17(13):4659.

33. Chen C-Y, McGee CW, Rich TL, Prudente CN, Gillick BT. Reference values of intrinsic muscle strength of the hand of adolescents and young adults. J Hand Ther Off J Am Soc Hand Ther. 2018 Sep;31(3):348–56.

34. Lam NW, Goh HT, Kamaruzzaman SB, Chin A-V, Poi PJH, Tan MP. Normative data for hand grip strength and key pinch strength, stratified by age and gender for a multiethnic Asian population. Singapore Med J. 2016 Oct;57(10):578–84.

35. Leyk D, Gorges W, Ridder D, Wunderlich M, Rüther T, Sievert A, et al. Hand-grip strength of young men, women and highly trained female athletes. Eur J Appl Physiol. 2007 Mar;99(4):415–21.

36. Martin JA, Ramsay J, Hughes C, Peters DM, Edwards MG. Age and grip strength predict hand dexterity in adults. PloS One. 2015;10(2):e0117598.

37. Timpka S, Petersson IF, Zhou C, Englund M. Muscle strength in adolescent men and risk of cardiovascular disease events and mortality in middle age: a prospective cohort study. BMC Med. 2014 Apr 14;12:62.

38. Ortega FB, Silventoinen K, Tynelius P, Rasmussen F. Muscular strength in male adolescents and premature death: cohort study of one million participants. BMJ. 2012 Nov 20;345:e7279.

39. European Food Safety Authority Panel on Dietetic Products, Nutrition and Allergies. Dietary reference values for vitamin D. EFSA Journal. 2016;14(10):4547 doi:10.2903/j.efsa.2016.4547.

40. McCarthy MS, Elshaw EB, Szekely BM, Raju D. A Prospective Cohort Study of Vitamin D Supplementation in AD Soldiers: Preliminary Findings. Mil Med. 2019 Mar 1;184(Suppl 1):498–505.

41. Pojednic RM, Ceglia L. The emerging biomolecular role of vitamin D in skeletal muscle. Exerc Sport Sci Rev. 2014 Apr;42(2):76–81.

42. Nimptsch K, Platz EA, Willett WC, Giovannucci E. Association between plasma 25-OH vitamin D and testosterone levels in men. Clin Endocrinol (Oxf). 2012 Jul;77(1):106–12.

43. Mielgo-Ayuso J, Calleja-González J, Urdampilleta A, León-Guereño P, Córdova A, Caballero-García A, et al. Effects of Vitamin D Supplementation on Haematological Values and Muscle Recovery in Elite Male Traditional Rowers. Nutrients [Internet]. 2018 Dec 12 ;10(12):1968.

44. Kraemer WJ, Ratamess NA, Hymer WC, Nindl BC, Fragala MS. Growth Hormone(s), Testosterone, Insulin-Like Growth Factors, and Cortisol: Roles and Integration for Cellular Development and Growth With Exercise. Front Endocrinol. 2020;11:33.

45. Duclos M, Gouarne C, Bonnemaison D. Acute and chronic effects of exercise on tissue sensitivity to glucocorticoids. J Appl Physiol Bethesda Md 1985. 2003 Mar;94(3):869–75.

46. Lombardi G, Vitale JA, Logoluso S, Logoluso G, Cocco N, Cocco G, et al. Circannual rhythm of plasmatic vitamin D levels and the association with markers of psychophysical stress in a cohort of Italian professional soccer players. Chronobiol Int. 2017;34(4):471–9.

47. Fitzgerald JS, Orysiak J, Wilson PB, Mazur-Różycka J, Obminski Z. Association between vitamin D status and testosterone and cortisol in ice hockey players. Biol Sport. 2018 Sep;35(3):207–13.

48. Wrzosek M, Woźniak J, Włodarek D. The Combination of a Diversified Intake of Carbohydrates and Fats and Supplementation of Vitamin D in a Diet Does Not Affect the Levels of Hormones (Testosterone, Estradiol, and Cortisol) in Men Practicing Strength Training for the Duration of 12 Weeks. Int J Environ Res Public Health. 2020 Nov 1;17(21).

49. Dodds RM, Syddall HE, Cooper R, Kuh D, Cooper C, Sayer AA. Global variation in grip strength: a systematic review and meta-analysis of normative data. Age Ageing. 2016 Mar;45(2):209–16.

50. Haslam A, Johnson MA, Hausman DB, Cress ME, Houston DK, Davey A, et al. Vitamin D status is associated with grip strength in centenarians. J Nutr Gerontol Geriatr. 2014;33(1):35–46.

51. Wang J, Wang X, Gu Y, Liu M, Chi VTQ, Zhang Q, et al. Vitamin D is related to handgrip strength in adult men aged 50 years and over: A population study from the TCLSIH cohort study. Clin Endocrinol (Oxf). 2019 May;90(5):753–65.

52. Granlund L, Norberg M, Ramnemark A, Andersson C, Lindkvist M, Fhärm E. Vitamin D is associated with lower limb muscle strength and grip strength in Middle Eastern- and African-born immigrants in Sweden. Nutr Res N Y N. 2018 Nov;59:29–35.

53. Wakayo T, Belachew T, Whiting SJ. Serum Vitamin D Level Associates With Handgrip Muscle Strength Among Ethiopian Schoolchildren: A Cross-Sectional Study. Food Nutr Bull. 2018 Mar;39(1):54–64.

54. von Hurst PR, Conlon C, Foskett A. Vitamin D status predicts hand-grip strength in young adult women living in Auckland, New Zealand. J Steroid Biochem Mol Biol. 2013 Jul;136:330–2.

55. Gaffney-Stomberg E, Nakayama AT, Guerriere KI, Lutz LJ, Walker LA, Staab JS, et al. Calcium and vitamin D supplementation and bone health in Marine recruits: Effect of season. Bone. 2019 Jun;123:224–33.

56. Willis KS, Peterson NJ, Larson-Meyer DE. Should we be concerned about the vitamin D status of athletes? Int J Sport Nutr Exerc Metab. 2008 Apr;18(2):204–24.

57. Allison RJ, Close GL, Farooq A, Riding NR, Salah O, Hamilton B, et al. Severely vitamin D-deficient athletes present smaller hearts than sufficient athletes. Eur J Prev Cardiol. 2015 Apr;22(4):535–42.

58. Hughes JM, Smith MA, Henning PC, Scofield DE, Spiering BA, Staab JS, et al. Bone formation is suppressed with multi-stressor military training. Eur J Appl Physiol. 2014 Nov;114(11):2251–9.

59. Goodman GP, Schoenfeld AJ, Owens BD, Dutton JR, Burks R, Belmont PJ. Non-emergent orthopaedic injuries sustained by soldiers in Operation Iraqi Freedom. J Bone Joint Surg Am. 2012 Apr 18;94(8):728–35.

60. Lappe J, Cullen D, Haynatzki G, Recker R, Ahlf R, Thompson K. Calcium and vitamin d supplementation decreases incidence of stress fractures in female navy recruits. J Bone Miner Res Off J Am Soc Bone Miner Res. 2008 May;23(5):741–9.

61. Aranow C. Vitamin D and the immune system. J Investig Med Off Publ Am Fed Clin Res. 2011 Aug;59(6):881–6.

62. Christensen MHE, Lien EA, Hustad S, Almås B. Seasonal and age-related differences in serum 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D and parathyroid hormone in patients from Western Norway. Scand J Clin Lab Invest. 2010 Jul;70(4):281–6.

63. Carmeliet G, Dermauw V, Bouillon R. Vitamin D signaling in calcium and bone homeostasis: a delicate balance. Best Pract Res Clin Endocrinol Metab. 2015 Aug;29(4):621–31.

64. Reid IR, Bolland MJ. Controversies in medicine: the role of calcium and vitamin D supplements in adults. Med J Aust. 2019;211(10):468–73.

65. Scott JM, Kazman JB, Palmer J, McClung JP, Gaffney-Stomberg E, Gasier HG. Effects of vitamin D supplementation on salivary immune responses during Marine Corps basic training. Scand J Med Sci Sports. 2019 Sep;29(9):1322–30.

66. Rips L, Toom A, Kull M, Varblane A, Gapejeva H, Kartus JT, Ööpik V, Tammaru M, Rahu M. Seven- month wintertime supplementation of 1200 IU vitamin D has no effect on hand grip strength in young, physically active males: a randomized, controlled study. https://datadoi.ee/handle/33/342, http://dx.doi.org/10.23673/re-284

Seven-month Wintertime Supplementation of 1200 IU Vitamin D Has No Effect on Hand Grip Strength in Young, Physically Active Males: A Randomized, Controlled Study.

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