MOTIVATION: Thermotolerance during prolonged exercise in the heat is associated with adaptations linked to the heat shock response. This response confers transient thermal tolerance, in part due to the expression of heat shock proteins (Hsps). Hsp72 and Hsp27 are particularly responsive to physiological stress, including hyperthermia. During exercise-heat stress, extracellular levels of Hsp72 are proposed to increase in conjunction with the level of hyperthermia attained, regardless of the rate of heat storage. However, exercise perfomed to exhaustion at different intensities in the heat may elicit differing rates of Hsp expression related to the development of thermal and metabolic strain. In addition, the association between Hsp72 and Hsp27 expression in the extracellular milieu remains largely unexplored. Therefore, this study examined the influence of exercise at moderate and high intensities on plasma Hsp72 and Hsp27 expression. METHODS: Sixteen male subjects cycled to exhaustion at 60% and 75% of maximal oxygen uptake (VO2max) in hot conditions (40°C, 50% relative humidity). Core temperature, heart rate, oxidative stress, blood lactate and blood glucose levels were measured during exercise to determine the predictor variables associated with plasma Hsp72 and Hsp27 concentrations prior to exercise, on reaching exhaustion, and 24 h following exercise cessation. RESULTS: At exhaustion, heart rate exceeded 96% of maximum in both moderate and high intensity exercise conditions. A core temperature of 39.7 ± 0.4°C was reached in the 60% VO2max trial after 58.9 ± 10.9 min of exercise, whereas 39.0 ± 0.5°C was attained in the 75% VO2max trial after 27.2 ± 9.0 min (mean ± SD; P < 0.001). In the 75% VO2max trial, the rate of rise in core temperature was 2.1 ± 1.4°C/h greater than in the 60% VO2max trial (P < 0.001). A significant increase and correlation was observed between plasma Hsp72 and Hsp27 concentrations at exhaustion (P < 0.005). Plasma Hsp72 was highly correlated with the core temperature attained in the 60% VO2max trial and the rate of increase in core temperature in the 75% VO2max trial (P < 0.05). However, no common predictor variable was associated with the expression of both Hsps. CONCLUSION: The similarity in plasma Hsp72 and Hsp27 concentration during moderate and high intensity exercise may relate to the duration (i.e. core temperature attained) and intensity (i.e. rate of increase in core temperature) of exercise. As such, the expression of plasma Hsp72 and Hsp27 in response to exercise in the heat appears to be duration and intensity dependent.