Gastrointestinal (GI) ischemia during exercise is associated with luminal permeability and increased systemic lipopolysaccharides (LPS). This study aimed to assess the impact of a multistrain pro/prebiotic/antioxidant intervention on endotoxin unit levels and GI permeability in recreational athletes. Thirty healthy participants (25 males, 5 females) were randomly assigned either a multistrain pro/prebiotic/antioxidant (LAB⁴_ANTI; 30 billion CFU·day^-1 containing 10 billion CFU·day^-1Lactobacillus acidophilus CUL-60 (NCIMB 30157), 10 billion CFU·day^-1Lactobacillus acidophillus CUL-21 (NCIMB 30156), 9.5 billion CFU·day^-1Bifidobacterium bifidum CUL-20 (NCIMB 30172) and 0.5 billion CFU·day^-1Bifidobacterium animalis subspecies lactis CUL-34 (NCIMB 30153)/55.8 mg·day^-1 fructooligosaccharides/ 400 mg·day^-1 α-lipoic acid, 600 mg·day^-1N-acetyl-carnitine); matched pro/prebiotic (LAB⁴) or placebo (PL) for 12 weeks preceding a long-distance triathlon. Plasma endotoxin units (via Limulus amebocyte lysate chromogenic quantification) and GI permeability (via 5 h urinary lactulose (L): mannitol (M) recovery) were assessed at baseline, pre-race and six days post-race. Endotoxin unit levels were not significantly different between groups at baseline (LAB⁴_ANTI: 8.20 ± 1.60 pg·mL^-1; LAB⁴: 8.92 ± 1.20 pg·mL^-1; PL: 9.72 ± 2.42 pg·mL^-1). The use of a 12-week LAB⁴_ANTI intervention significantly reduced endotoxin units both pre-race (4.37 ± 0.51 pg·mL^-1) and six days post-race (5.18 ± 0.57 pg·mL^-1; p = 0.03, ηp² = 0.35), but only six days post-race with LAB⁴ (5.01 ± 0.28 pg·mL^-1; p = 0.01, ηp² = 0.43). In contrast, endotoxin units remained unchanged with PL. L:M significantly increased from 0.01 ± 0.01 at baseline to 0.06 ± 0.01 with PL only (p = 0.004, ηp² = 0.51). Mean race times (h:min:s) were not statistically different between groups despite faster times with both pro/prebiotoic groups (LAB⁴_ANTI: 13:17:07 ± 0:34:48; LAB⁴: 12:47:13 ± 0:25:06; PL: 14:12:51 ± 0:29:54; p > 0.05). Combined multistrain pro/prebiotic use may reduce endotoxin unit levels, with LAB⁴_ANTI potentially conferring an additive effect via combined GI modulation and antioxidant protection.

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