Introduction: Functional testing of isolated mitochondria is a powerful in vitro tool for determining mitochondrial health following an ischemic stress. Successful preservation of intact mitochondria requires an efficient harvest technique under cold conditions. As heart and brain mitochondria are vital to the success of cardiopulmonary resuscitation (CPR), we sought to determine the consequence of co-isolation of these organs on cardiac mitochondrial function in a porcine model of cardiac arrest. Methods: After 15 min of ventricular fibrillation, 36 animals were randomized to active compression decompression + impedance threshold device (ACD-ITD) CPR (Control), or ACD-ITD CPR + ischemic postconditioning (IPC) and further randomized to heart-only (Control n=9, IPC n=11) or heart + brain mitochondrial isolation (Control n=8, IPC n=8). IPC consisted of 3 cycles of 20 sec compression / 20 sec pause for the first 2 min of CPR. Cardiac and brain mitochondria were isolated via differential centrifugation 4 min after initiation of CPR. To minimize warm ischemia, brain biopsies were performed while animals were supine with uninterrupted chest compressions. Heart biopsies were subsequently performed via intercostal incision. Respiratory control index (RCI) and calcium retention capacity (CRC) were measured for complex I and II substrates. Results: Due to technique, heart isolation was delayed by 100 s in heart + brain vs. heart-only animals. In animals with heart-only isolation, IPC increased complex I and II RCI and complex I CRC compared to Controls. In animals with heart + brain isolation, cardiac complex I and II RCI and complex I CRC decreased within the IPC group but not within the Control group. There were no differences in brain mitochondrial function between IPC and Controls. Conclusion: Mitochondrial isolation from multiple organs must consider warm ischemia that may worsen mitochondrial performance. Communication between the brain and heart during biopsy appears to be mediated by a mechanism antagonistic to the beneficial effects of IPC, as isolation of heart and brain resulted in deleterious cardiac mitochondrial performance in IPC but not in Controls. Further investigation is warranted to refine multi-organ mitochondrial isolation.