Wildland firefighting requires repetitive (e.g., consecutive work shifts) physical work in dangerous conditions (e.g., heat and pollution). Workers commonly enter these environments in a nonacclimated state, leading to fatigue and heightened injury risk. Strategies to improve tolerance to these stressors are lacking. Purpose: To determine if glutamine ingestion prior to and after consecutive days of firefighting simulations in the heat attenuates subjective ratings of fatigue, and evaluate if results were supported by glutamine-induced upregulation of biological stress responses. Methods: Participants (5 male, 3 female) ingested glutamine (0.15 g/kg/day) or a placebo before and after two consecutive days (separated by 24 h) of firefighter simulations in a heated chamber (35 °C, 35% humidity). Perceived fatigue and biological stress were measured pre-, post-, and 4 h postexercise in each trial. Results: Subjective fatigue was reduced pre-exercise on Day 2 in the glutamine group (p < 0.05). Peripheral mononuclear cell expression of heat shock protein 70 (HSP70) and serum antioxidants were elevated at 4 h postexercise on Day 1 in the glutamine trial (p < 0.05). Conclusions: Ingestion of glutamine before and after repeated firefighter simulations in the heat resulted in reduced subjective fatigue on Day 2, which may be a result of the upregulation of biological stress systems (antioxidants, HSPs). This response may support recovery and improve work performance.