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This randomized controlled trial aimed to test whether vitamin D (VD) supplementation affects measures of physical performance in VD-deficient, mildly trained children. Thirty-six recreationally soccer player boys were randomly assigned to single dose (200 000 IU) of VD3 (n = 19) or placebo (n = 17). Plasma 25-hydroxyvitamin D (25-OHD) was assessed and measures of physical performance (i.e., vertical and standing broad jumps, triple hop, 10-m and 30-m sprints, shuttle run) were performed before and 12 weeks after the loading dose. Mixed ANCOVA models were performed and effect size was estimated by partial eta squared (ηp2). Baseline 25-OHD and physical variables were equivalent in the 2 groups. Twelve weeks after VD loading, plasma 25-OHD increased and physical variables improved only in the VD group. There was a significant interaction effects for group by time for vertical jump (F = 14.9, p = 0.001, ηp2 = 0.394), triple hop jump (F = 24.2, p < 0.001, ηp2 = 0.513), 10-m (F = 4.46, p = 0.046, ηp2 = 0.162) and 30-m (F = 6.56, p = 0.017, ηp2 = 0.222) sprints, and shuttle run (F = 13.4, p = 0.001, ηp2 = 0.369). In conclusion, a single bolus of VD3 resulted in significant improvements in jumping ability, agility, and running speed in mildly trained children that are deficient in VD. The findings suggest that correcting VD deficit might be beneficial for physical performance. Novelty A mega dose of VD3 improves jumping ability, agility, and running speed in VD-deficient, mildly trained children. Effect of VD on measures of physical performance is noticeable 3 months after the loading dose.