Introduction: Athletes engaged in repeated-sprint training in the heat can be at an increased risk of gastrointestinal ischemia and damage in response to a redistribution of blood to working skeletal muscles and the skin. This study investigated the effects of repeated sprinting in hot and cool conditions on markers of gastrointestinal damage. Methods: Twenty-five, well-trained, nonheat acclimated male team-sport athletes completed a five-session, repeated-sprint training regimen over 7 days in either HOT (40 °C and 40% relative humidity [RH]) or COOL (20 °C and 40% RH) conditions. Participants underwent a 20-min warm-up and four sets of 5 × 6-s maximal cycling sprints, with 24-s rest and 5-min recovery between sets. Venous blood was collected pre-, post-, and 1 hr postexercise and analyzed for intestinal fatty acid binding protein, lipopolysaccharide binding protein, soluble CD14, and heat-shock protein. Results: Intestinal fatty acid binding protein concentrations were significantly increased (p < .004) postexercise (593 and 454 pg/ml) and 1 hr postexercise (466 and 410 pg/ml) on both Days 1 and 5 in HOT. Soluble CD14 increased by 398 and 308 ng/ml postexercise (p = .041), and lipopolysaccharide binding protein increased by 1,694 ng/ml postexercise on Day 1 in HOT (p < .05) and by 1,520 ng/ml on Day 5 in COOL (p = .026). Core and skin temperature, rating of perceived exertion, and thermal sensation were higher (p < .05) in HOT on Days 1 and 5 during sprinting. Conclusions: Repeated sprinting in the heat induced greater thermal strain and mild changes in gastrointestinal damage, likely attributable to the combination of environmental conditions and maximal-intensity exercise.