AbstractIncreased gut permeability is implicated in the initiation and extent of the cytokine inflammatory response associated with exertional heat stroke (EHS). The primary objective of this study was to determine if a five amino acid oral rehydration solution (5AAS), specifically designed for the protection of the gastrointestinal lining, would prolong time to EHS, maintain gut function and dampen the systemic inflammatory response (SIR) measured during EHS recovery. Male C57/BL6J mice instrumented with radiotelemetry were gavaged with 150 μL of 5AAS or H2O, and ≈12 h later were either exposed to an EHS protocol where mice exercised in a 37.5°C environmental chamber to a self‐limiting maximum core temperature (Tc,max) or performed the exercise control (EXC) protocol (25°C). 5AAS pretreatment attenuated hypothermia depth and length (p< 0.005), which are indicators of EHS severity during recovery, without any effect on physical performance or thermoregulatory responses in the heat as determined by percent body weight lost (≈9%), max speed (≈6 m/min), distance (≈700 m), time to Tc,max (≈160 min), thermal area (≈550°C∙min), and Tc,max (42.2°C). EHS groups treated with 5AAS showed a significant decrease in gut transepithelial conductance, decreased paracellular permeability, increased villus height, increased electrolyte absorption and changes in tight junction protein expression pattern suggestive of improved barrier integrity (p< 0.05). No differences were witnessed between EHS groups in acute phase response markers of liver, circulating SIR markers, or indicators of organ damage during recovery. These results suggest that a 5AAS improves Tc regulation during EHS recovery through maintaining mucosal function and integrity.