The purpose of this study was to determine gastrointestinal (GI) permeability during prolonged treadmill running (60 min at 70 % V·O2max) with and without fluid intake (3 ml/kg body mass/10 min). Twenty runners (11 males, 9 females; age = 22 ± 3 (SD) yrs; mean V·O2max = 55.7 ± 5.0 ml/kg/min) completed four experiments: 1) rest, 2) running with no fluid (NF), 3) running with ingestion of a 4 % glucose solution (GLU), and 4) running with ingestion of a water placebo (PLA). To determine GI permeability, subjects also drank a solution containing 5 g sucrose (S), 5 g lactulose (L), and 2 g rhamnose (R) immediately prior to each trial. Gastroduodenal permeability was determined by urinary S excretion, while small intestinal permeability was determined by the L/R excretion ratio. Percent body mass loss (i.e., dehydration) was negligible during rest, GLU and PLA, while NF resulted in a 1.5 % loss of body mass (p < 0.05). Gastroduodenal and intestinal permeability were significantly (p < 0.008) increased in NF compared to rest. There were no other differences in GI permeability. These results indicate that fluid restriction during 1 h of steady-state running increases GI permeability above resting levels.