The objective of this study was to determine if prolonged exercise resulted in the appearance of cardiac troponin T (cTnT) in serum and whether this was associated with elevated levels of myocardial oxidative stress. Forty-five male Sprague-Dawley rats were randomized into four groups and killed before (PRE-EX), immediately (0HR), 2 (2HR) and 24 h (24HR) after a 3-h bout of swimming with 5% body weight attached to their tail. In all animals serum cTnT was assayed using 3rd generation electrochemiluminescence. In homogenized heart tissue myocardial malondialdehyde (MDA), a marker of lipid peroxidation, glutathione (GSH), and a non-enzymatic estimate of total antioxidant capacity (T-AOC) were assessed spectrophotometrically. At PRE-EX cTnT was undetectable in all animals. At 0HR (median, range: 0.055, 0.020-0.100) and 2HR post-exercise (0.036, 0.016-2.110) cTnT was detectable in all animals (P < 0.05). At 24HR post-exercise cTnT was undetectable in all animals. An elevation in MDA was observed 0HR (mean ± SD: 1.7 ± 0.2 nmol mgpro(-1)) and 2HR (1.6 ± 0.3 nmol mgpro(-1)) post-exercise compared with PRE-EX (1.3 ± 0.2 nmol mgpro(-1); P < 0.05). The antioxidant response to this challenge was a significant (P < 0.05) decrease in GSH 2HR and 24HR post-exercise. Despite this T-AOC did not alter across the trial (P > 0.05). The results indicated that prolonged and strenuous exercise in rats resulted in an elevation in cTnT, a biomarker of cardiomyocyte damage, in all animals 0HR and 2HR after exercise completion. The time course of cTnT elevation was temporally associated with evidence of increased lipid peroxidation in the rat heart.