PURPOSE: The study aimed to determine the effect of diurnal versus nocturnal exercise on gastrointestinal integrity and functional responses, plasma lipopolysaccharide binding protein (LBP) and soluble CD14 (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% relative humidity) running, on one occasion performed at 0900 h (400 lx; DAY) and on another occasion at 2100 h (2 lx; NIGHT). Blood samples were collected pre- and postexercise 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 determined by lactulose challenge test given at 150 min, with concomitant breath hydrogen (H2) and gastrointestinal symptom determination. RESULTS: Cortisol increased substantially pre- to postexercise 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 postexercise on both trials (mean = 2269 pg·mL-1, 95% confidence interval = 1351-3187, +143%) (main effect of time [MEOT], P < 0.001). sCD14 increased pre- to postexercise (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 180 min postexercise) compared with DAY (mean = 54 min, 95% confidence interval = 29-79). NIGHT resulted in greater total gastrointestinal symptoms (P = 0.009) compared with DAY. No difference in feeding tolerance markers was observed between trials. CONCLUSION: Nocturnal exercise instigates greater gastrointestinal functional perturbations and symptoms compared with diurnal exercise. However, there are no circadian differences to gastrointestinal integrity and systemic perturbations in response to the same exertional stress and controlled procedures.