INTRODUCTION: It is not clear whether the levels of troponin I (TI) and creatine kinase isoenzyme MB (CK-MB) are changed in premature infants (PI) without evidence of myocardial ischemia (MI). OBJECTIVES: To investigate whether TI and CK-MB change their levels in newborns without MI while on mechanical ventilation (MV). METHODS: We conducted a prospective cohort study in which 165 PI were divided into control group ([CG]; n = 68), mechanical ventilated group ([VG]; n = 21) and a surfactant therapy group ([SG]; n = 76), and had their TI, creatine kinase (CK) and CK-MB levels were determined. After the division, within the first four hours after the introduction of the mechanical ventilation (MV) and one hour after the withdrawal of it, we performed a new measurement of TI, CK and CK-MB to all PI from VG and VS. We used the chi-square test to evaluate the association among qualitative variables and the Kruskall-Wallis test to compare the serum levels of TI, and CK-MB among the groups, before, during and after MV using the statistical package SPSS 16.0 software. RESULTS: TI, CK, and CK-MB serum values before the groups were divided were considered normal. The TI concentration among the groups before and after MV (p > 0.05) did not changed; however, the CK-MB levels were higher in VG when compared to the CG (p = 0.009). CONCLUSION: The increase of CK-MB serum levels in VG and SG seems to indicate an increased work of thoracic skeletal muscle and do not represent a MI signal, which invalidate its use as a marker.