Clinically and experimentally, a case for omega-3 polyunsaturated fatty acid (PUFA) cardioprotection in females has not been clearly established. The goal of this study was to investigate whether dietary omega-3 PUFA supplementation could provide ischemic protection in female mice with an underlying genetic predisposition to cardiac hypertrophy. Mature female transgenic mice (TG) with cardiac specific over-expression of angiotensinogen which develop normotensive cardiac hypertrophy, and littermate wild type (WT) mice were fed a fish oil-derived diet (FO) or PUFA-matched control diet (CTR) for 4 weeks. Myocardial membrane lipids, ex vivo cardiac performance (intraventricular balloon) after global no-flow ischemia and reperfusion (15/30min), and reperfusion arrhythmia incidence, were assessed. FO diet suppressed cardiac growth by 5% and 10%, in WT and TG respectively (P<0.001). The extent of mechanical recovery (rate pressure product= bpm x mmHg) of FO-fed WT and TG hearts was similar (50±7% vs. 45±12%, 30min reperfusion) and this was not significantly different from CTR-fed WT or TG. To evaluate whether systemic estrogen was masking a protective effect of the FO diet, the responses of ovariectomized (OVX) WT and TG mice to FO dietary intervention was assessed. The extent of mechanical recovery, of FO-fed OVX WT and TG (RPP, 50±4% vs 64±8%) was not enhanced compared with CTR-fed (RPP, 60±11% vs 80±8% P=0.335). Dietary FO did not suppress the incidence of reperfusion arrhythmias in WT or TG hearts (ovary intact or OVX). Our findings indicate a lack of cardioprotective effect of dietary FO in females, determined by assessment of mechanical and arrhythmic activity post-ischemia in a murine ex vivo heart model.