PURPOSE: To test the potential for the ex situ limb perfusion system to prolong limb allograft survival up to 24 hours. METHODS: We used 20 swine for the study. In group 1 (control), 4 limbs were perfused with heparin solution and preserved at 4°C for 6 hours. In group 2, 4 limbs were perfused with autologous blood at 27°C to 32°C for 24 hours. In both groups, limbs were transplanted orthotopically to recipients and monitored for 12 hours. In addition to perfusion parameters, we recorded perfusate gases and electrolytes (pH, pCO2, pO2, O2 saturation, Na, K, Cl, Ca, HCO3, glucose, and lactate) and obtained functional electrostimulation hourly throughout the experiment. Histology samples were obtained for TUNEL staining and single-muscle fiber contractility testing. RESULTS: In both groups, hemodynamic variables of circulation remained stable throughout the experiment. Neuromuscular electrical stimulation remained intact until the end of reperfusion in group 2 vs no response in group 1. In group 2, a gradual increase in lactate levels during pump perfusion returned to normal after transplantation. Compared with the contralateral limb in group 2, single-muscle fiber contractility testing showed no significant difference at the end of the experiment. CONCLUSIONS: We demonstrated extended limb survival up to 24 hours using normothermic pulsatile perfusion and autologous blood. CLINICAL RELEVANCE: Successful prolongation of limb survival using ex situ perfusion methods provides with more time for revascularization of an extremity.