This study aimed to examine the effects of a multi-day mountain bike race on amateur master athlete cyclists (AMA). AMA (50±5 years) completed 2 stages of a mountain bike race. Six weeks before racing, echocardiography, blood and maximal cardiopulmonary exercise tests (CPET) were performed. One hour after completion of each race stage, echocardiography measurements were taken and blood was sampled for stress, inflammatory and cardiac biomarkers. Main outcomes of the CPET were maximal oxygen consumption (VO2max) 50.7±6.5 ml/kg/min; maximal power 328±45 Watt and ventilatory threshold of 86%VO2max. Participants completed 95.7km with an elevation of 1650m on the first day, and 92.5km with an elevation of 1410m on the second day, with completion times of 357±42 and 390 ±43 minutes, respectively. After racing, increases in Creatine Phosphokinase and C-reactive protein (3-6 fold) (p<0.001), Troponin I (4-fold) (p<0.001) and N-terminal pro-brain natriuretic peptide (NT-Pro BNP) (4-7-fold) (p<0.001) were noted, with a reduction in the myocardial global work index of 17-24% (p<0.001). No correlations were found between Troponin I or NT-Pro BNP and myocardial global work index. Highly aerobically fit AMA participating in demanding endurance events demonstrated elevated stress, inflammatory, muscle damage and cardiac biomarkers. However, these changes did not significantly correlate with altered cardiac function. In addition, consecutive days of demanding prolonged cycling exercise did not have cumulative effects on the measured myocardial parameters.