Observational studies have raised concerns that prolonged strenuous exercise training may be associated with increased risk of cardiac arrhythmia and even primary cardiac arrest or sudden death. It has been demonstrated that leucine can reduce prolonged exercise-induced muscle damage and accelerate the recovery process. The aim of this study was to investigate the effects of prolonged strenuous endurance exercise on cardiovascular parameters and biomarkers of cardiac injury in trained adult male rats and assess the use of leucine as an auxiliary substance to prevent the likely cardiac adverse effects caused by strenuous exercise. Twenty-four male Wistar rats were randomly allocated to receive a balanced control diet (18% protein) or a leucine-rich diet (15% protein plus 3% leucine) for 6 weeks. The rats were submitted to 1 h of exercise, 5 days per week for 6 weeks. Three days after the training period, the rats were submitted to swimming exercise until exhaustion, and cardiac parameters were assessed. Exercising until exhaustion significantly increased cardiac biomarker levels, cytokines and glycogen content inhibited protein synthesis signalling and led to cardiac electrical disturbances. When combined with exercise, leucine supplementation led to greater increases in the aforementioned parameters and also a significant increase in blood pressure and protein degradation signalling. We report, for the first time, that leucine supplementation not only fails to prevent cardiac fatigue symptoms, but may also aggravate prolonged strenuous exercise-induced cardiovascular disturbances in trained rats. Furthermore, we find that exercising until exhaustion can cause cardiac electrical disturbances and damage cardiac myocytes.