Protection against post-infarction myocardial dysfunction is known to be rather modest with classical preconditioning. The aim of our study was to investigate whether multiple cycles-preconditioning could improve this protection and whether post-infarction dysfunction only depends on the amount of viable tissue. Accordingly, 18 rabbits were chronically instrumented with coronary balloon occluders and ultrasonic crystals in the ischemic zone. Ten days later, a Control group of conscious animals underwent a 30min coronary artery occlusion (CAO) followed by 72h reperfusion (CAR). In two other groups, preconditioning (PC) was previously induced either by six 4min-CAO/4min-CAR cycles (PCx6) or by one 5min-CAO/10min-CAR cycle (PCx1). After 72h-CAR, functional recovery reached a plateau in all groups. Depression in segment shortening was reduced in PCx1 (-68±7% from baseline) and to a greater extent with PCx6 (-18±10%) vs Control (-99±7%; all p<0.05). Infarct sizes were reduced in PCx1 (15±2%) and to a greater extent with PCx6 (3±1%) vs Control (46±5%; p<0.05). The contractility of salvaged myocardium was evaluated by calculating the ratio between segment shortening at 72h-CAR and the amount of viable tissue. This index was significantly enhanced in PCx1 (0.39±0.07) and to a greater extent in PCx6 (0.82±0.09) vs Control (0.0±0.10). Immunohistochemistry demonstrated that this differential effect of PC was not related to changes in apoptosis, eNOS expression or macrophages infiltration but rather to blunted endothelial iNOS expression in PCx6 vs Control and PCx1. In conclusion, we demonstrated that multiple cycles-preconditioning induced an almost complete protection against post-infarction dysfunction, potentially involving beneficial effects on salvaged myocardium.