The aim of this study was to determine the clinical relevance of cardiac biomarkers [troponin I and T, creatine kinase-MB fraction (CK-MB) and lactate dehydrogenase (LDH)] in premature calves with respiratory distress syndrome. Seventy premature calves were admitted to the clinic within 24 h after birth. Respiratory distress syndrome was diagnosed in premature calves by clinical examination and venous blood gas analysis. Ten healthy calves, aged 5 days, were used as control. Cardiac troponin I and T were analysed using ELISA and ELFA, respectively. Serum CK-MB and LDH were also analysed in an automatic analyser. The calves had low venous pH, pO2, O2 saturation and high pCO2 values consistent with dyspnoea, hypoxaemia, and inadequate oxygen delivery. Mean serum troponin I, troponin T, CK-MB and LDH levels were increased in the premature calves compared to the control group. In conclusion, the results in this study demonstrated that serum CK-MB, troponin I and troponin T concentrations could be used for evaluating myocardial injury in premature calves with respiratory distress syndrome.