It is commonly believed that gastrointestinal issues during exercise are exacerbated by hypohydration. This study aimed to determine the effect of exercise-induced hypohydration on gastrointestinal integrity, function, symptoms, and systemic endotoxin and inflammatory profiles. In a randomized crossover design, male endurance runners (n 11) performed 2 h of running at 70% of maximum oxygen uptake in 25°C ambient temperature with water provision [euhydration (EuH)] and total water restriction [hypohydration (HypoH)] during running, which accounted for 0.6 0.6% and 3.1 0.7% body mass loss, respectively. Blood and fecal samples were collected before and after exercise. Breath samples (H2 determination) were collected and gastrointestinal symptoms (GIS) recorded before, during, and after exercise. HypoH resulted in a higher, yet insignificant, preexercise to postexercise plasma cortisol concentration (286 nmol/l vs. 176 nmol/l; P 0.098) but significantly higher intestinal fatty acid-binding protein (I-FABP) (539 pg/ml vs. 371 pg/ml; P 0.047) concentration compared with EuH. A greater breath H2 response (P 0.026) was observed on HypoH (1,188 ppm/3 h, peak 12 ppm) vs. EuH (579 ppm/3 h, peak 6 ppm). Despite greater GIS incidence on HypoH (82%) vs. EuH (64%), GIS severity scores were not significant between trials. Exercise-induced leukocytosis (overall pre- to postexercise: 5.9 109/l to 12.1 109/l) was similar on both trials. Depressed in vitro neutrophil function was observed during recovery on HypoH (36%) but not on EUH (6%). A pre- to postexercise increase (P 0.05) was observed for circulating cytokine concentrations but not endotoxin values. Hypohydration during 2 h of running modestly perturbs gastrointestinal integrity and function and increases GIS incidence but does not affect systemic endotoxemia and cytokinemia. NEW & NOTEWORTHY Despite anecdotal beliefs that exercise-induced hypohydration exacerbates perturbations to gastrointestinal status, the present study reports only modest perturbations in gastrointestinal integrity, function, and symptoms compared with euhydration maintenance. Exercise-induced hypohydration does not exacerbate systemic endotoxemia and cytokinemia compared with euhydration maintenance. Programmed water intake to maintain euhydration results in gastrointestinal symptom severity similar to exercise-induced hypohydration. Maintaining euhydration during exertional stress prevents the exercise-associated depression in bacterially stimulated neutrophil function.