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?°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?°C resulted in significantly increased oesophageal (39.3?±?0.2?°C) and muscle temperature (40.0?±?0.2?°C) at the end of the exercise protocol compared with 20?°C (oesophageal, 38.1?±?0.1?°C; muscle, 38.9?±?0.2?°C). However, an increased DNA binding of HSF was not evident following exercise at 40?°C (reduced by 21?±?22%) whereas it increased by 29?±?51% following exercise at 20?°C. Conclusion: It appears that increased temperature is not the major factor responsible for activation of HSF DNA binding.