Skeletal muscle is highly adaptable in response to changes in contractile activity. The purpose of this study was to determine if skeletal muscle preconditioning exerted a protective effect against subsequent denervation-induced apoptotic protein expression. To investigate this we chronically stimulated the tibialis anterior (TA) and extensor digitorum longus (EDL) muscles for 7 days (10 Hz, 3 hrs/ day) prior to 7 days of denervation. Denervation reduced cytochrome c oxidase (COX) activity by approximately 40%, and this was attenuated by prior chronic stimulation. Denervation increased the expression of Bax by 2.2-fold, and prior chronic stimulation did not alter this increase. This produced an elevation in the Bax:Bcl-2 ratio, indicating greater muscle apoptotic susceptibility. Denervation reduced total muscle mass by 25-30%, an effect which was not influenced by prior chronic stimulation. Denervation also decreased state 3 respiration in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria, and elevated state 4 ROS production within both mitochondrial subfractions. These changes were not prevented by prior chronic stimulation, despite a stimulation-induced increase in the anti-oxidant enzyme MnSOD. The autophagic regulatory protein Beclin-1 was markedly elevated by denervation, suggesting that autophagic cell death could also play a role in denervation-induced muscle atrophy. Thus, despite prior chronic stimulation which elevated mitochondrial content, subsequent denervation reversed this increase, and augmented the apoptotic susceptibility of muscle by altering the Bax:Bcl-2 ratio and increasing ROS production. Whether a more extensive paradigm of chronic contractile activity would be more effective in attenuating apoptotic protein expression prior to muscle disuse remains to be determined.