We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of 3 cycles of 10 min occlusion/reperfusion in a hind limb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hind limb remote IPC also induces a late phase of infarct protection in skeletal muscle, and K_ATP channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 ±2% infarction when subjected to 4h of ischemia/48h of reperfusion. The late phase of infarct protection appeared at 24h and lasted up to 72h after hind limb remote IPC. The LD muscle infarction was reduced to 28 ± 3, 26 ± 1, 23 ± 2, 24 ±2 and 24 ± 4% at 24, 28, 36, 48 and 72h after remote IPC, respectively (p < 0.05; n=8). In subsequent studies, hind limb remote IPC or intravenous injection of the sarcolemmal K_ATP (sK_ATP) channel opener P-1075 (2 µg/kg) at 24h before 4h of sustained ischemia (i.e. late preconditioning) reduced muscle infarction from 43 ± 4% (ischemic control) to 24 ± 2 and 19 ± 3%, respectively (p < 0.05, n=8). Intravenous injection of the sK_ATP channel inhibitor HMR 1098 (10 mg/kg) or the non-specific K_ATP channel inhibitor glibenclamide (Glib, 1 mg/kg) at 10 min before remote IPC completely blocked the infarct protective effect of remote IPC in LD muscle flaps subjected to 4h of sustained ischemia at 24h after remote IPC. Intravenous bolus injection of the mitochondrial K_ATP (mK_ATP) channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg) immediately before remote IPC and 30 min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD but not HMR 1098 given 24h after remote IPC completely blocked the late infarct protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (p<0.05) muscle content of ATP at the end of 4h of ischemia and 1.5h of reperfusion and a lower (p<0.05) neutrophilic activity at the end of 1.5h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hind limb remote IPC induces an uninterrupted long (48h) late phase of infarct protection, and sK_ATP and mK_ATP channels play a central role in the trigger and mediator mechanism, respectively.