Regular physical exercise can positively influence cardiac function; however, investigations have shown an increase of myocardial damage biomarkers after acute prolonged endurance exercises. We investigated the effect of repeated sprint vs. moderate long duration exercise on markers of myocardial necrosis, as well as cardiac dimensions and functions. Thirteen healthy males performed two different running sessions (randomized, single blinded cross-over design): 60 minutes moderate intensity continuous training (MCT, at 70% of peak heart rate (HRpeak)) and two series of 12 × 30-second sprints with set recovery periods in-between (RST, at 90% HRpeak). Venous blood samples for cardiac troponin T (cTnT), creatine kinase (CK) and MB isoenzyme (CK-MB) were taken 1 and 4 hours after exercise sessions. After each session electrocardiographic (ECG) and transthoracic echocardiographic (TTE) data were recorded. Results showed that all variables - average heart rate, serum lactate concentration during RST, subjective exertion and cTnT after RST - were significantly higher compared to MCT. CK and CK-MB significantly increased regardless of exercise protocol, while ECG and TTE indicated normal cardiac function. Our results provide evidence that RST contributes significantly to cTnT and CK release. This biomarker increase seems to reflect a physiological rather than a pathological phenomenon in healthy, exercising subjects.