Skeletal muscle adapts to the stress of acute (Morton et al., 2006) and chronic (Morton et al., in press) exercise via increased content of heat shock proteins (HSPs) and antioxidant defences. An increased content of such defences following stress function to restore cellular homeostasis and to protect the cell from further insults. A consistent elevation of ‘stress proteins’ during training may therefore be a crucial component of the cellular mechanisms by which regular exercise provides protection against exercise-induced muscle damage. Adaptations of HSPs and antioxidants following running exercise have only been studied in the vastus lateralis, despite the gastrocnemius muscle being more metabolically active in this exercise mode(Costill et al., 1974). The aim of this study was to compare HSP and antioxidant adaptations in the vastus lateralis and gastrocnemius following high intensity interval training. Eight males performed 50 min of high intensity intermittent running exercise, four times per week for six weeks. The protocol consisted of five 3 min bouts at 90% 2max separated by 3 min active recovery periods (1.5 min at 25% 2max followed by 1.5 min at 50% 2max). A 10 min warm-up and cool down period at 70% 2max was also performed. Assessments of 2max, running economy (RE) and performance on a Yo-Yo intermittent recovery 2 test (IRT2) were performed before and after training. Resting muscle biopsies were obtained from the lateral portion of the gastrocnemius and from the mid-portion of the vastus lateralis muscle pre- and post-training. Training induced significant (P<0.05) improvements in 2max (10%), RE (4%) and IRT2 (16%). Training resulted in significant increases (P<0.05) in HSP60 (22%), αB-crystallin (52%), MnSOD (38%) and a tendency (P<0.09) for an increase in HSP70 (20%) content of the gastrocnemius. In contrast, only αB-crystallin (22%) and MnSOD (37%) significantly increased (P<0.05) in the vastus lateralis following training. HSP27 was unchanged in either muscle following training. This study is the first to examine HSP and antioxidant protein adaptations of the gastrocnemius and vastus lateralis muscle to exercise. Data demonstrate that short-term high intensity intermittent exercise induces a more pronounced up-regulation of stress proteins in the gastrocnemius muscle compared with the vastus lateralis. This differential response between muscles is likely due to differences in recruitment patterns (and hence greater activation of associated signalling pathways) during this mode of exercise. We therefore suggest that the gastrocnemius muscle is a more suitable muscle for which to study the exercise-induced stress response of human skeletal muscle during running.