Background: Cardioplegic solutions assure only a sub-optimal myocardial protection during prolonged storage for transplantation. The ultimate cause of myocardial damage during storage is unknown, but oxygen free radicals might be involved. We evaluated the occurrence of oxidative stress and changes in cellular redox potential after different periods of hypothermic storage. Methods Langendorff-perfused rabbit hearts were subjected to a protocol mimicking each stage of a cardiac transplantation procedure: explantation, storage and reperfusion. Three periods of storage were considered: Group A = 5 hours, Group B = 15 hours, and Group C = 24 hours. Oxidative stress was determined in terms of myocardial content and release of reduced (GSH) and oxidized (GSSG) glutathione, and cellular redox potential as oxidized and reduced pyridine nucleotides ratio (NAD/NADH). Data on mechanical function, cellular integrity and myocardial energetic status were collected. Results At the end of reperfusion, despite the different timings of storage, recovery of left ventricular developed pressure (46.1 ± 7.0, 54.7 ± 6.7, and 45.7 ± 7.4% of the baseline pre-ischaemic value), energy charge (0.81 ± 0.02, 0.81 ± 0.02, and 0.77 ± 0.01) and NAD/NADH ratio (8.87 ± 1.08, 9.39 ± 1.72, and 10.26 ± 1.98) were similar in all groups (A, B and C). On the contrary, the rise in left ventricular resting pressure (LVRP) and GSH/GSSG ratio were significantly different between Group C, and Groups A and B ( p < 0.0001, analyzed by Generalized Estimating Equations model for repeated measures, and p < 0.05, respectively). Conclusions The pathophysiology of myocardial damage during hypothermic storage cannot be considered as a normothermic ischaemic injury and parameters other than energetic metabolism, such as thiolic redox state, are more predictive of functional recovery upon reperfusion.