AIM: The present study investigated whether increased activation of heat shock factors (HSF) following exercise relates primarily to the increased muscle temperature or to exercise in general. METHODS: Six subjects completed 40 min of intermittent cycling (15s:15s exercise:recovery at 300 +/- 22 W) at an ambient temperature of either 20.0 +/- 1.3 or 40.3 +/- 0.7 degrees C. Muscle biopsies were taken prior to and immediately following the exercise protocol with samples analysed for HSF DNA binding by electrophoretic mobility shift assay. RESULTS: Exercise at 40 degrees C resulted in significantly increased oesophageal (39.3 +/- 0.2 degrees C) and muscle temperature (40.0 +/- 0.2 degrees C) at the end of the exercise protocol compared with 20 degrees C (oesophageal, 38.1 +/- 0.1 degrees C; muscle, 38.9 +/- 0.2 degrees C). However, an increased DNA binding of HSF was not evident following exercise at 40 degrees C (reduced by 21 +/- 22%) whereas it increased by 29 +/- 51% following exercise at 20 degrees C. CONCLUSION: It appears that increased temperature is not the major factor responsible for activation of HSF DNA binding.