Background/Objectives: Vitamin D plays an important role in muscle metabolism and recovery, yet its kinetics during and after football-specific physical activity remain poorly understood. Therefore, this study aimed to determine whether physical effort during a football match influences the concentration of vitamin D metabolites and to explore the effect of a single high-dose cholecalciferol supplementation combined with physical exercise on the levels of vitamin D metabolites in professional football players. Methods: Twenty professional football players participated in a three-phase, randomized placebo-controlled pilot study. Baseline fitness and blood samples were collected, followed by pre- and post-match measurements during two games. In the final phase, half of the players received a single 500,000 IU dose of vitamin D3 before a simulated match. Blood samples were collected before and after each session to analyze vitamin D metabolites using the isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method. Results: Physical exercise during the football match significantly increased serum concentrations of 25-(OH)D3, 24,25-(OH)2D3, and 3-epi-25-(OH)D3 (by up to 25%, p < 0.001). Following supplementation, these effects were further amplified, with 25-(OH)D3 rising by 98% and 3-epi-25-(OH)D3 by 424% (p < 0.001). Significant alterations in vitamin D metabolite ratios after exercise and supplementation suggest enhanced metabolic turnover and dynamic regulation of vitamin D pathways in response to physical effort. Conclusions: Football-specific physical activity appears to stimulate the release of vitamin D metabolites. High-dose cholecalciferol supplementation was well tolerated and may rapidly increase vitamin D status in professional athletes. These findings may have implications for optimizing recovery and performance, though larger trials are needed.
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