Abstract
This study investigated the effects of an 8-week daily 2000 IU vitamin D₃ supplementation, administered via orodispersible films, on serum vitamin D, leukocyte count, and performance parameters in healthy recreational runners and non-runners, followed by a 12-week post-supplementation follow-up. Forty-five participants were randomized into four groups: supplemented runners (RUN-SUPPL, n = 13, age 33.4 ± 7.5 y), non-supplemented runners (RUN-CON, n = 10, age 34.8 ± 8.6 y), supplemented non-runners (NON-RUN-SUPPL, n = 11, age 29.3 ± 7.5 y), and non-supplemented non-runners (NON-RUN-CON, n = 11, age 28.3 ± 6.0 y). Assessments were conducted at baseline (T0, October, pre-supplementation), post-intervention (T1, December, after 8 weeks), and follow-up (T2, March, 12-weeks post-supplementation). Supplementation significantly increased serum 25(OH)D₃ in runners (30.45 ± 7.0 to 35.35 ± 8.9 ng/mL, p < 0.001) and non-runners (25.0 ± 8.7 to 30.2 ± 9.7 ng/mL, p < 0.001), while non-supplemented non-runners showed a marked decline (25.93 ± 6.6 to 17.8 ± 7.3 ng/mL, p < 0.001). After follow-up, serum vitamin D decreased significantly in all groups (e.g., RUN-SUPPL 23.52 ± 5.6 ng/mL, p < 0.001). A significant time-by-supplementation interaction was observed for total leukocytes (p = 0.001) and neutrophils (p = 0.03), indicating more stable immune profiles in supplemented participants. No significant changes were found in VO₂max or countermovement jump (p > 0.05), while maximal isometric force showed a trend toward improvement in supplemented subjects (p = 0.056). Despite these physiological benefits, no ergogenic effects on aerobic or explosive performance were observed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-026-38528-1.