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Purpose: This study aimed to investigate the effects of multi-strain probiotics supplementation on gastrointestinal permeability, systemic markers of inflammation and running performance when exercising in the heat., Methods: Ten male runners were randomized to 4 weeks of daily supplementation with a probiotics capsule (45 billion CFU of Lactobacillus, Bifidobacterium and Streptococcus strains) or placebo, separated by a washout period (double-blind, cross-over trial). After each treatment, the runners exercised to fatigue at 80% of their ventilatory threshold at 35 °C and 40% humidity. To assess gastrointestinal permeability, runners ingested lactulose and rhamnose before exercise and post-exercise urine was collected to measure sugar concentrations. Venous blood samples were collected before, immediately after and 1 h after exercise, and core temperature was monitored during exercise., Results: Probiotics supplementation significantly increased run time to fatigue (min:s 37:44 ± 2:42 versus 33:00 ± 2:27; P = 0.03, d = 0.54). Average core temperature during exercise was similar between trials (probiotic 38.1 ± 0.2 °C, placebo 38.1 ± 0.1 °C; P = 0.77, d = 0.13). Serum lipopolysaccharide concentration increased post-exercise (P < 0.001), while there was a moderate to large reduction in pre-exercise (d = 0.70) and post-exercise (d = 1.24) concentration following probiotics supplementation. Plasma concentrations of IL-6, IL-10 and IL-1ra increased after exercise (P < 0.01), but there was no significant difference between trials (P > 0.05). There was a small to moderate reduction (d = 0.35) in urine lactulose:rhamnose and a small reduction (d = 0.25) in symptoms of gastrointestinal discomfort following probiotics supplementation (both P = 0.25)., Conclusion: Four weeks of supplementation with a multi-strain probiotic increased running time to fatigue in the heat. Further studies are required to elucidate the exact mechanisms for this performance benefit.