Simple SummaryVitamin D3may regulate inflammation, immunological responses, and muscle damage; however, its impact on these physiological activities remains obscure. This study reported the effects of vitamin D3supplementation on immune response, inflammatory profile, muscle damage, and aerobic capacity. The participants received either 5000 IU of vitamin D3or placebo for 4 weeks. The time to exhaustion, maximal heart rate, and average heart rate were also recorded during the exercise test. Venous blood samples were drawn before, immediately after, and 2, 4, and 24 h after exercise to assess total and differential blood leukocyte counts, levels of cytokines, and muscle damage biomarkers. We found that daily 5000 IU vitamin D3supplementation for 4 weeks may effectively increase blood 25(OH)D levels, immune response, and aerobic capacity while inhibiting inflammatory cytokines and muscle damage in people performing strenuous endurance exercise. Vitamin D3seems to be a useful nutritional strategy to attenuate immune suppression, inflammatory responses, and muscle damage in people performing an intensive exercise.AbstractReportedly, strenuous endurance exercise can depress the immune system and induce inflammation and muscle damage. Therefore, this double-blinded, matched-pair study aimed to investigate the impact of vitamin D3supplementation on immune response (leukocyte, neutrophil, lymphocyte, CD4+, CD8+, CD19+, and CD56+counts), inflammatory profile (TNF-α and IL-6), muscle damage (CK and LDH levels), as well as aerobic capacity after strenuous endurance exercise in 18 healthy men taking 5000 IU of vitamin D3(n= 9) or placebo (n= 9) daily for 4 weeks. Total and differential blood leukocyte counts, levels of cytokines, and muscle damage biomarkers were determined before, immediately after, and 2, 4, and 24 h after exercise. The IL-6, CK, and LDH levels were significantly lower in vitamin D3group at 2, 4, and 24 h post exercise (p< 0.05). Maximal and average heart rates during exercise were also significantly lower (p< 0.05). In the vitamin D3group, the CD4+/CD8+ratio after 4 weeks of supplementation was only significantly lower at post-0 than at baseline and significantly higher at post-2 than at baseline and post-0 (allp< 0.05). Taken together, 5000 IU of daily vitamin D3supplementation for 4 weeks exhibited positive effects in terms of increased blood 25(OH)D levels, CD4+/CD8+ratio (immune response), and aerobic capacity while inhibiting inflammatory cytokines and CK and LDH (muscle damage) in people performing strenuous endurance exercise.