Purpose The study aimed to determine the impact of diurnal versus nocturnal exercise on gastrointestinal integrity and functional responses, plasma LBP and sCD14 concentrations (as indirect indicators of endotoxin responses), systemic inflammatory cytokine profile, gastrointestinal symptoms and feeding tolerance. Methods Endurance runners (n=16) completed 3 h of 60% V˙O2max (22.7°C, 45% RH) running, on one occasion performed at 0900h (400-lux; DAY) and another occasion at 2100h (2-lux; NIGHT). Blood samples were collected pre- and post-exercise, and during recovery to determine plasma concentrations of cortisol, catecholamines, claudin-3, I-FABP, LBP and sCD14, and inflammatory cytokine profiles by ELISA. Orocecal transit time (OCTT) was determine by lactulose challenge test given at 150 min, with concomitant breath hydrogen (H2) and gastrointestinal symptoms (GIS) determination. Results Cortisol increased substantially pre- to post-exercise on NIGHT (+182%) versus DAY (+4%) (trial×time, p=0.046), with no epinephrine (+41%) and norepinephrine (+102%) trial differences. I-FABP, but not claudin-3, increased pre- to post-exercise on both trials (2269 (1351-3187) pg·ml-1; +143%) (main effect of time (MEOT), p<0.001). sCD14 increased pre- to post-exercise (trial×time, p=0.045; +5.6%), and was greater on DAY; but LBP decreased (MEOT, p=0.019, -11.2%) on both trials. No trial difference was observed for systemic cytokine profile (MEOT, p=0.004). Breath H2 responses (p=0.019) showed that OCTT was significantly delayed on NIGHT (>84 min, with n=3 showing no breath H2 turning point by 180min post-exercise) compared to DAY (54 (29-79) min). NIGHT resulted in greater total-GIS (p=0.009) compared to DAY. No difference in feeding tolerance markers was observed between trials. Conclusion Nocturnal exercise instigates greater gastrointestinal functional perturbations and symptoms compared to diurnal exercise. Although there are no circadian differences to gastrointestinal integrity and systemic perturbations in response to the same exertional stress and controlled procedures.