Purpose: The study aimed to determine the effects of exertional-heat stress on gastrointestinal integrity, symptoms, systemic endotoxin and inflammatory responses; and assess the relationship between changes in body temperature and gastrointestinal perturbations. Methods: Ten endurance runners completed 2 h running at 60% V˙ O2max in hot (HOT: 35 °C) and temperate (TEMP: 22 °C)-ambient conditions. Rectal temperature (Tre) and gastrointestinal symptoms were recorded every 10 min during exercise. Blood samples were collected pre- and post-exercise, and during recovery to determine plasma intestinal fatty acid binding protein (I-FABP), cortisol, bacterial endotoxin and cytokine profile. Calprotectin was determined from pre- and post-exercise faecal samples. Urinary lactulose:l-rhamnose ratio was used to measure intestinal permeability. Results: Compared with TEMP, HOT significantly increased Tre (1.4 ± 0.5 vs 2.4 ± 0.8 °C, p < 0.001), cortisol (26 vs 82%, p < 0.001), I-FABP (127 vs 432%, p < 0.001), incidence (70 vs 90%) and severity (58 counts vs 720 counts, p = 0.008) of total gastrointestinal symptoms. Faecal calprotectin and circulating endotoxin increased post-exercise in both trials (mean increase 1.5 ± 2.5 µg/g, p = 0.032, and 6.9 ± 10.3 pg/ml, p = 0.047, respectively), while anti-endotoxin antibodies increased 28% post-exercise in TEMP and decreased 21% in HOT (p = 0.027). However, intestinal permeability did not differ between trials (p = 0.185). Inflammatory cytokines were greater on HOT compared to TEMP (p < 0.05). Increases in Tre were positively associated with I-FABP, IL-10, cortisol, nausea and urge to regurgitate (p < 0.05). Conclusions: Exertional-heat stress induces a thermoregulatory strain that subsequently injures the intestinal epithelium, reduces endotoxin clearance capacity, promotes greater cytokinaemia, and development of gastrointestinal symptoms.