Currently, controversial clinical data about the protective effects in the consumption of n-3 polyunsaturated fatty acids (PUFAs) in ischaemic heart diseases exist. Improved myocardial resistance to ischaemia-reperfusion (IR) injury results in non-lethal myocardial infarction, which is a relevant factor in the myocardial function. We hypothesized that chronic supplementation with PUFAs reduced infarct size (IS) and induced an improvement on oxidative stress-related parameters in IR model. Rats were supplemented with two doses of PUFAs D1 (n=7) (0.6gkg(-1)d(-1)) and D2 (n=7) (1.2gkg(-1)d(-1)) for 8weeks. Control group (n=7) received only standard diet. In ex vivo model, all rat hearts were subjected to 30min of global ischaemia followed by 120min of reperfusion. The IS and left ventricular function were assessed. Lipid peroxidation, reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio and antioxidant enzyme activity were measured in the whole heart. The results show a reduction in IS in a dose-dependent manner with PUFAs D1 (30.6%) and D2 (48.5%) and higher values of left ventricular developed pressure, at the end of the reperfusion, for each dose, respectively (p<0.05). The two PUFAs groups showed higher values of GSH/GSSG ratio and lipid peroxidation, and higher values of activity of antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase at basal condition (p<0.05). At the end of reperfusion, the GSH/GSSG ratio and antioxidants enzyme activity did not show a significant drop in their values (p>0.05). These findings suggested that the supplementation with PUFAs induces cardioprotection against IR injury, associated with reinforcement of the antioxidant defense system. Copyright (c) 2013 John Wiley & Sons, Ltd.