Introduction Decreased physical fitness and inferior physical performance are risk factors and potentially life threatening in the combat situation. To examine the effect of Vitamin D on physical performance a prospective longitudinal 10-month study of young male conscripts in the Estonian Army was designed. The hypothesis of the study was, that severe deficiency of Vitamin D has a negative effect on physical performance. Materials All conscripts (n = 410) entering the 10-month military service in July 2015 at the Kuperjanov Battalion were asked to participate. A total of 98 male conscripts volunteered to participate initially. Study design and data collection A prospective longitudinal study with a 10-month follow-up period was performed. The Army Physical Fitness Test (APFT) test was performed three times, the hand grip strength and blood serum values of 25(OH)D, parathyroid hormone (PTH) and calcium (Ca) were measured four times. Results A significant decrease in the levels of 25(OH)D compared with the baseline values were found, with a lowest mean value of 31.9 nmol/l in March 2016 (p 25nmol/L. Discussion The main finding of the present study was that severe deficiency of 25(OH)D were common among male conscripts during the winter season, but had no negative effect on physical performance in terms of the APFT test and hand grip strength test. Conclusion Severe deficiency of Vitamin D during the winter season are common but have no negative effect on physical performance in young physically active men in military service.