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We sought to determine the effects of probiotic supplementation (Bacillus subtilis DE111; 1 billion CFU∙d⁻¹) on markers of immune and hormonal status in collegiate male athletes following 12 weeks of offseason training. Twenty-five Division I male baseball athletes (20.1 ± 1.5 years, 85.5 ± 10.5 kg, 184.7 ± 6.3 cm) participated in this double blind, placebo-controlled, randomized study. Participants were randomly assigned to a probiotic (PRO; n = 13) or placebo (PL; n = 12) group. Pre- and post-training, all athletes provided resting blood and saliva samples. Circulating concentrations of testosterone, cortisol, TNF-α, IL-10, and zonulin were examined in the blood, while salivary immunoglobulin A (SIgA) and SIgM were assayed as indicators of mucosal immunity. Separate analyses of covariance (ANCOVA) were performed on all measures collected post intervention. No differences in measures of body composition or physical performance were seen between groups. TNF-α concentrations were significantly (p = 0.024) lower in PRO compared to PL, while there were no significant group differences in any other biochemical markers examined. A main effect for time was observed (p < 0.05) for increased testosterone (p = 0.045), IL-10 (p = 0.048), SIgA rate (p = 0.031), and SIgM rate (p = 0.002) following offseason training. These data indicate that probiotic supplementation had no effect on body composition, performance, hormonal status, or gut permeability, while it may attenuate circulating TNF-α in athletes.