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To investigate the effects of dietary creatine supplementation alone and in combination with exercise on basal cardiac function, susceptibility to ischaemia/reperfusion injury and mitochondrial oxidative function. There has been an increase in the use of creatine supplementation among sports enthusiasts, and by clinicians as a therapeutic agent in muscular and neurological diseases. The effects of creatine have been studied extensively in skeletal muscle, but not in the myocardium.Male Wistar rats were swim-trained for 8 weeks, 5 days per week. Hearts were excised and either freeze-clamped for biochemical analysis or perfused on the isolated heart perfusion system to assess function and ischaemia/reperfusion tolerance. Mechanical function was documented in working heart and retrograde mode. The left coronary artery was ligated and infarct size determined. Mitochondrial oxidative capacity was quantified.Aortic output recovery of hearts from the sedentary controls (CSed) was significantly higher than those from creatine-supplemented sedentary (CrSed), creatine-supplemented exercised (CrEx) as well as control exercised (CEx) groups. Ischaemic contracture of hearts from CrEx was significantly higher than that of CSed. There were no differences in infarct size and mitochondrial oxygen consumption.This study suggests that creatine supplementation has no effects on basal cardiac function but reduces myocardial tolerance to ischaemia in hearts from exercise-trained animals, by increasing the ischaemic contracture and decreasing reperfusion aortic output. Exercise training alone also significantly decreased aortic output recovery. However, the exact mechanisms for these adverse myocardial effects are unknown and need further investigation.