To examine the effect of exercise on heat shock protein (HSP) 72 mRNA expression in skeletal muscle, five healthy humans (20 ± 1 yr; 64 ± 3 kg; peak O2 uptake of 2.55 ± 0.2 l/min) cycled until exhaustion at a workload corresponding to 63% peak O2uptake. Muscle was sampled from the vastus lateralis, and muscle temperature was measured at rest (R), 10 min of exercise (Min10), ∼40 min before fatigue (F-40 = 144 ± 7 min), and fatigue (F = 186 ± 15 min). Muscle samples were analyzed for HSP72 mRNA expression, as well as glycogen and lactate concentration. Muscle temperature increased ( P < 0.05) during the first 10 min of exercise but then remained constant for the duration of the exercise. Similarly, lactate concentration increased ( P < 0.05) when Min10 was compared with R but decreased ( P < 0.05) thereafter, such that concentrations at F-40 and F were not different from those at R. In contrast, muscle glycogen concentration fell progressively throughout exercise (486 ± 74 vs. 25 ± 7 mmol/kg dry weight for R and F, respectively; P < 0.05). HSP72 mRNA was detected at R but did not increase by Min10. However, HSP72 mRNA increased ( P < 0.05) 2.2 ± 0.5- and 2.6 ± 0.9-fold, respectively, when F-40 and F were compared with R. These data demonstrate that HSP72 mRNA increases progressively during acute cycling, suggesting that processes that take place throughout concentric exercise are capable of initiating a stress response.