Oxidative stress occurs during ex-situ heart perfusion (ESHP) and may negatively affect functional preservation of the heart. We sought to assess the status of key antioxidant enzymes during ESHP, and the effects of augmenting these antioxidants on the attenuation of oxidative stress and improvement of myocardial and endothelial preservation in ESHP. Porcine hearts were perfused for 6 hours with oxygen-derived free-radical scavengers polyethylene glycol (PEG)-catalase or PEG-superoxide dismutase (SOD) or with naive perfusate (control). The oxidative stress-related modifications were determined in the myocardium and coronary vasculature, and contractile function, injury, and endothelial integrity were compared between the groups. The activity of key antioxidant enzymes decreased and adding catalase and SOD restored the enzyme activity. Cardiac function and endothelial integrity were preserved better with restored catalase activity. Catalase and SOD both decreased myocardial injury and catalase reduced ROS production and oxidative modification of proteins in the myocardium and coronary vasculature. The activity of antioxidant enzymes decrease in ESHP. Catalase may improve the preservation of cardiac function and endothelial integrity during ESHP. While catalase and SOD may both exert cardioprotective effects, unbalanced SOD and catalase activity may paradoxically increase the production of reactive species during ESHP.