This randomized, single-blind, placebo-controlled pilot study investigated the effects of high-dose vitamin D₃ supplementation and football-specific physical exercise on vitamin D metabolite concentrations in professional male football players. The study aimed to evaluate the acute response of circulating vitamin D metabolites, including 25-(OH)D₃, 24,25-(OH)₂D₃, and 3-epi-25-(OH)D₃, following a single oral dose of 500,000 IU cholecalciferol combined with high-intensity intermittent exercise typical of competitive football.

Twenty professional football players from a Polish top-division club participated in the study. Participants were randomly assigned to either the supplementation group (SGP) receiving 500,000 IU of vitamin D₃ or the placebo group (PGP) receiving an identical volume of placebo oil. The intervention was administered 48 hours prior to an intra-squad game. Blood samples were collected at three time points: baseline (T3), pre-match (T4), and post-match (T5). The primary outcome was the change in serum 25-(OH)D₃ concentration. Secondary outcomes included changes in 24,25-(OH)₂D₃ and 3-epi-25-(OH)D₃ levels, as well as their ratios. The study also assessed whether physical exercise modulates vitamin D metabolism.

The findings demonstrated that high-dose vitamin D₃ supplementation significantly increased serum 25-(OH)D₃ (↑198%) and 3-epi-25-(OH)D₃ (↑444%) levels, while football-specific exercise itself induced moderate increases in vitamin D metabolites. The results suggest that skeletal muscle may play a key role in vitamin D storage and release in response to exercise. The supplementation was well tolerated, with no adverse events observed.

This pilot study was designed to explore the combined effects of high-dose vitamin D₃ supplementation and acute physical exercise on vitamin D metabolism in professional football players. The study responds to growing scientific interest in understanding how vitamin D status influences performance, recovery, and muscle function in athletes. While most previous studies have focused on chronic, low-to-moderate supplementation, this trial examined the response to a single, pharmacological dose of cholecalciferol in a controlled experimental setting.

Study Design

The study employed a randomized, single-blind, placebo-controlled, parallel-group design. Participants were randomly assigned to one of two groups:

Supplementation Group (SGP) - received a single oral dose of 500,000 IU vitamin D₃ (Vigantol Oil, P&G Health, Germany);

Placebo Group (PGP) - received an identical amount of placebo (vegetable oil).

The supplementation was administered 48 hours before a standardized intra-squad football match (ISG), conducted under conditions simulating a competitive game. All players followed the same training schedule and diet during the study.

Participants

A total of 20 professional male football players (aged 18-35) from a Polish top-league club volunteered to participate. Inclusion criteria included active professional status, regular training participation, and no use of vitamin D or calcium supplements in the previous 3 months. Exclusion criteria included musculoskeletal injuries, metabolic disorders, or refusal to sign informed consent.

Measurements

Blood samples were collected at three time points:

T3: baseline (before supplementation),

T4: 48 hours post-supplementation (pre-match),

T5: immediately after the intra-squad game.

Serum concentrations of 25-(OH)D₃, 24,25-(OH)₂D₃, and 3-epi-25-(OH)D₃ were determined using LC-MS/MS. Additional biochemical variables (hemoglobin, glucose, testosterone, cortisol, ferritin, calcium, CK) were analyzed to monitor physiological status.

Outcomes

Primary Outcome: Changes in serum 25-(OH)D₃ concentrations after supplementation and exercise.

Secondary Outcomes: Changes in 24,25-(OH)₂D₃ and 3-epi-25-(OH)D₃ levels, metabolite ratios.

Ethics

The study was approved by the Ethics Committee of the Wroclaw University of Health and Sport Sciences (Resolution No. 1/2024, March 1, 2024). All participants signed written informed consent.

Inclusion Criteria:

1. Male professional football players aged 18-35 years
2. Actively training and competing in the Polish first football league
3. No vitamin D or calcium supplementation during the previous 3 months
4. No injuries or illnesses affecting participation

Exclusion Criteria:

1. Current musculoskeletal injury or illness
2. Chronic metabolic, endocrine, or renal disorders
3. Use of vitamin D or calcium supplements
4. Refusal or inability to sign informed consent

Individual participant data will not be shared due to privacy considerations and the specific nature of the study population (professional athletes). Aggregated data are available upon reasonable request to the corresponding author.

Single-blind design: participants were unaware of whether they received vitamin D₃ or placebo; the investigator administering supplements was unblinded.