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Introduction: Deficient levels of 25-hydroxyvitamin D (25(OH)D) (<30 ng/mL) may compromise health and athletic performance. Supplementation with oral vitamin D can favor the state of iron metabolism, and testosterone and cortisol as an indicator of muscle recovery of the athlete with a deficiency. The main aim of this study was to evaluate the influence of eight weeks of supplementation with 3000 IU/day of vitamin D on the hematological and iron metabolism profile, as well as on the analytical values of testosterone and cortisol on elite male traditional rowers. The secondary aim was to examine if serum 25(OH)D is a predictor of testosterone and cortisol levels. Material and Methods: Thirty-six elite male rowers (27 ± 6 years) were assigned to one of the two groups randomly: 1) Control group (CG,= 18, height: 181.05 ± 3.39 cm and body mass: 77.02 ± 7.55 kg), 2) Group treated with 3,000 IU of vitamin D3/day (VD3G,= 18, height: 179.70 ± 9.07 cm and body mass: 76.19 ± 10.07 kg). The rowers were subjected to blood tests at the beginning of the study (T1) and after eight weeks of treatment (T2), for the analysis of hematological and hormonal values. Repeated-measures ANOVA with group factor (GC and GVD3) were used to examine if the interaction of the different values was the same or different between the groups throughout the study (time × group) after vitamin D3 treatment. To analyze if 25(OH)D was a good predictor of testosterone, cortisol, and testosterone/cortisol ratio a stepwise regression model was performed. Results: Statistically significant and different increases were observed in the group-by-time interaction of 25(OH)D in VD3G in respect to CG during the study (< 0.001; VD3G (T1: 26.24 ± 8.18 ng/mL vs. T2: 48.12 ± 10.88 ng/mL) vs CG (T1: 30.76 ± 6.95 ng/mL vs. T2: 35.14 ± 7.96 ng/mL). Likewise, significant differences between groups were observed throughout the study in the group-by-time interaction and changes of hemoglobin (GC: −2.89 ± 2.29% vs. VD3G: 0.71 ± 1.91%;= 0.009), hematocrit (CG: −1.57 ± 2.49% vs. VD3G: 1.16 ± 1.81%;= 0.019) and transferrin (CG: 0.67 ± 4.88% vs. VD3G: 6.51 ± 4.36%;= 0.007). However, no differences between groups were observed in the group-by-time interaction of the hormonal parameters (> 0.05). Regression multivariate analysis showed that cortisol and testosterone levels were associated with 25(OH)D levels (< 0.05). Conclusion: Oral supplementation with 3000 IU/day of vitamin D3 during eight weeks showed to be sufficient to prevent a decline in hematological levels of hemoglobin and hematocrit, and improve transferrin of 25(OH)D levels. However, although it was not sufficient to enhance muscle recovery observed by testosterone and cortisol responses, it was observed that serum 25(OH)D levels could be a predictor of anabolic and catabolic hormones. View Full-Text