Abstract: The aim of this study was to evaluate the effect of preexercise glycerol hyperhydration on endurance performance in a heat chamber designed to simulate the World Championship Distance (WCD) duathlon (10km run, 40km ride, 5 km run). Duathlons are often performed in hot and humid conditions and as a result hydration is a major issue. Glycerol enhances the body’s capacity for fluid retention by inducing hyperhydration, which is theorized to improve thermoregulatory and cardiovascular responses, and thereby improve performance. Six well-trained athletes completed the testing protocol in a heat chamber at the La Trobe University Exercise Physiology Laboratory. Each testing session was approximately 4.5 hours in duration (2 hours of pre-exercise glycerol hyper-hydration followed by approximately 2.5 hours of exercise). The results showed an increased water retention pre-exercise and an improved overall performance of 2.04% was achieved by subjects ingesting the glycerol solution.