Different exercise protocols lead to long-term adaptations that are related to increased mitochondrial content through the activation of mitochondrial biogenesis. However, immediate mitochondrial response to exercise and energetic substrate utilization is still unknown. We evaluate the mitochondrial physiology of two types rat skeletal muscle fibres immediately after a single session of high intensity interval exercise (HIIE) or aerobic exercise (AER). We found AER was able to reduce the ATP synthesis dependent oxygen flux in the tibialis (TA) when stimulated by complex I and II substrates. On the other hand, there was an increase of the maximum velocity (V) for glycerol-phosphate oxidation and V and affinity (K) of palmitoyl-carnitine oxidation (PC). The exercise did not affect oxygen flux coupled to ATP synthesis in red gastrocnemius (RG) but, surprisingly, reduced its affinity for PC, decreasing the apparent catalytic efficiency (V/K) of oxidation for PC. Neither exercise protocol was able to change the electron transfer system capacity of the mitochondria or markers of mitochondrial content. The AER group had increased HO production compared to the SED and HIIE groups, with the mechanism being predominantly the escape of electrons through reverse flux in complex I and other sites in TA, and only through other sites in RG. There were no changes in the activities of antioxidant enzymes. Our results show that mitochondria from different muscles submitted to distinct exercise protocols show alterations in the specific fluxes of substrate utilization and oxygen metabolism, indicating that the dynamics of mitochondria are linked to the metabolic flexibility.