The measurement of cardiac troponins has emerged as the biochemical "gold standard" for the diagnosis and management of patients with acute chest pain. However, earlier markers should support investigation strategies, as several patients with acute coronary syndrome might present with non-diagnostic concentrations. Ischemia-modified albumin (IMA), measured by the albumin cobalt binding (ACB) assay, was recently proposed for early detection of myocardial ischemia. To establish the potential influence of endurance training on the diagnostic approach to patients with suspected myocardial injury, cardiac troponin T (cTnT), creatine kinase isoenzyme MB (CK-MB), myoglobin and IMA were evaluated in healthy individuals subjected to different aerobic workloads. The concentrations of both IMA and CK-MB were significantly increased in athletes subjected to high-workload endurance training, whereas the concentration of cTnT and myoglobin was not influenced by physical exercise in the medium term. Taken together, our results demonstrate that demanding aerobic physical activity might influence the generation of IMA, which might be increased in the medium term following high-workload endurance training, while the concentration of other conventional markers of myocardial injury remains non-diagnostic.