Effect of exercise on intestinal barrier function. (A) Serum 4-kDa FITC-Dextran concentration 1 h after oral gavage following 2, 4 and 6 weeks of exercise training. Data are presented as mean ± SEM; n = 9 (VIG-EX of Week 6) or n = 10 (other groups) per group. Statistical differences were analysed by one-way ANOVA followed by Bonferroni’s multiple comparison test. (B) Immunofluorescence staining of colonic occludin protein after 6 weeks of exercise training. The results were acquired by Leica TCS SP8 confocal microscope with HCX IRAPO L 25×/0.95 objective lens at 2.25×digital magnification; pinhole: 1.50 AU. Red colour: fluorescent signals from Alexa-Fluor 594 secondary antibodies. (C, D, E) Relative protein expression of colonic claudin-3, occludin and ZO-1 after 6 weeks of exercise training, assessed by Western blot. All target proteins were normalized to reference protein β-actin. Data are presented as mean ± SEM; n = 9-10 per group. Statistical differences were analysed by one-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01. CON: Control; MOD-EX: moderate exercise; VIG-EX: vigorous exercise; l.s.: longitudinal section; t.s.: transverse section; OCLN: occludin; ZO-1: zonula occludens-1; CLDN3: claudin-3.

The effect of exercise on the intestinal heat stress response. (A) Weekly post-exercise (after 15 min) rectal temperatures of mice acquired by a rectal thermometer. The dotted red line represents the average rectal temperature of all groups. Data are presented as mean ± SEM; n = 20 per group. Statistical differences were analysed by repeated measures two-way ANOVA. #### p < 0.0001 vs Control. Relative protein expression of (B) HSF-1 and (C) HSP70 in the colon after 6 weeks of exercise training assessed by Western blot. The target proteins were normalized to reference protein β-actin. Data are presented as mean ± SEM; n = 10 per group. Statistical differences were analysed by one-way ANOVA followed by Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001. CON: Control; MOD-EX: moderate exercise; VIG-EX: vigorous exercise; HSP: heat shock protein; HSF: heat shock factor.

Effect of exercise training on intestinal morphology and inflammation. (A) Representative images of H&E-stained proximal small intestinal (duodenal), distal small intestinal (ileal), and colonic sections of mice after 6 weeks of exercise training illustrate the inflammatory cell infiltration, epithelial changes and altered mucosa architecture. The images were acquired by Olympus BX50 microscope with UPlanFI 20× /0.25 objective lens and Leica DFC320 10× camera (200×, scale bar 200 μm). Solid arrow: mucosal or submucosal infiltration of leukocytes; dashed arrow: submucosal oedema; arrowhead: haemorrhage; Uc: ulceration; asterisk: blunted villus; Sw: submucosal widening. (B) Histological scores of intestinal morphology based on inflammatory cell infiltration, epithelial changes and altered mucosa architecture. (C) Representative images of cross-sectioned duodenal villi (D) Villus lengths measured by ImageJ software. (E) Percentages of villus goblet cells counted after Alcian Blue/Nuclear Fast Red staining. Data are presented as mean ± SEM; n = 10 mice per group. Statistical differences were analysed by one-way ANOVA followed by the Bonferroni’s multiple comparison test. ** p < 0.01, *** p < 0.001, **** p < 0.0001. CON: Control; MOD-EX: moderate exercise; VIG-EX: vigorous exercise.

The effect of exercise on caecal metabolite levels. Concentrations of short-chain fatty acids (A - F), lactate (G), and amino acids (H - K) in caecal contents after 6 weeks of exercise training measured 1 h after the last training bout by nuclear magnetic resonance spectroscopy. (L, M) Concentrations of total short-chain fatty acids and total amino acids in caecal contents after 6 weeks of exercise training measured 1 h after the last training bout. (N, O) The effect of strenuous exercise on colonic G-protein-coupled receptors 41 and 43. Relative protein expression of GPR41 and GPR43 in the murine colon after 6 weeks of exercise assessed by Western blot. All target proteins were normalized to reference protein β-actin. Data in the bar plots are presented as mean ± SEM; n = 10 per group. Statistical differences were analysed by one-way ANOVA followed by the Bonferroni’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CC: caecal contents; CON: Control; MOD-EX: moderate exercise; VIG-EX: vigorous exercise; SCFAs: short-chain fatty acids; AAs: amino acids; GPR: G-protein-coupled receptor.

The effect of exercise training on caecal microbiota diversity and composition. (A) Alpha diversity depicted as Chao1 index. (B) Beta diversity depicted as NMDS plot based on Bray–Curtis dissimilarity matrix. Each point represents a single sample and the closed areas represent confidence ellipses. The statistical significance among the groups is determined with permutational multivariate analysis of variance (PERMANOVA), adjusted for exercise status. (C, D) Bacterial taxonomic composition in the mouse cecum following 6 weeks of exercise training at phylum and family levels. The relative abundances are displayed as mean of samples. “Other” in (D) includes all the families with less than 0.2% abundance and unidentified families. (E, F, G) Pearson’s correlation analyses between the specific gut microorganism abundance and serum FITC-dextran concentration of CON, MOD-EX and VIG-EX. n = 9 (CON) or 10 (MOD-EX and VIG-EX). CON: Control; MOD-EX: moderate exercise; VIG-EX: vigorous exercise; NMDS: non-metric multi-dimensional scaling; AC : abundance counts.

Postulated mechanism underlying the systemic and local changes in the gastrointestinal tract induced by strenuous exercise. Solid or dashed arrow: induce, produce, activate or lead to; +/-: increase/decrease; bar-headed arrow: suppress. SCFAs: short-chain fatty acids; AAs: amino acids; GPR: G-protein coupled receptor.