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Reportedly, 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 D₃ supplementation 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 (<i>n</i> = 9) or placebo (<i>n</i> = 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 D₃ group at 2, 4, and 24 h post exercise (<i>p</i> < 0.05). Maximal and average heart rates during exercise were also significantly lower (<i>p</i> < 0.05). In the vitamin D₃ group, 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 (all <i>p</i> < 0.05). Taken together, 5000 IU of daily vitamin D₃ supplementation 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.