Ischemic tolerance decreases with ageing and the cardioprotective effect of ischemic preconditioning (IPC) is impaired in middle-aged animals. We have demonstrated that short-term caloric restriction (CR) improves myocardial ischemic tolerance in young and old animals via the activation of adiponectin-AMP activated kinase (AMPK)-mediated signaling. However, it is unknown whether prolonged CR confers cardioprotection in a similar manner. Furthermore, little is known regarding the myocardial expression of sirt1 (which reportedly mediates various aspects of the CR response) with prolonged CR. Thus, 6-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR groups. Six months later, isolated perfused hearts were subjected to 25 min of global ischemia followed by 120 min of reperfusion with or without IPC. CR improved the recovery of LV function and reduced infarct size after ischemia/reperfusion and restored the IPC effect. Serum adiponectin levels increased but myocardial levels of total and phosphorylated AMPK did not change with prolonged CR. Total levels of sirt1 did not change with CR; however, in the nuclear fraction, CR significantly increased sirt1 and decreased acetyl-histone H3. Eight rats from each group were given L-NAME in the drinking water for 4 weeks before sacrifice. In these hearts, chronic inhibition of nitric oxide synthase prevented the increase in nuclear sirt1 content by CR and abrogated CR-induced cardioprotection. These results demonstrate that (1) prolonged CR improves myocardial ischemic tolerance and restores the IPC effect in middle-aged rats, and that (2) CR-induced cardioprotection is associated with nitric oxide-dependent increase in nuclear sirt1 content.