Prolonged strenuous exercise is associated with the appearance of biomarkers of cardiac cell damage and a decline in cardiac function during recovery. Few studies have assessed repeated bouts of prolonged exercise and whether this results in further biomarker accumulation and greater dysfunction. Further, it may be useful to describe the changes in a range of biomarkers that may provide additional insight into the clinical significance of cardiac biomarker release. Four highly trained cyclists completed the 4800 km Race Across America (RAAM) in 7 days. Venous blood samples and echocardiograms were taken prior to, every 24 hours during and immediately after the RAAM. Venous blood was analysed for cardiac troponin I (cTnI), creatine kinase MB (CK-MB), fatty acid binding protein (HFABP), glycogen phosphorylase BB (GPBB) and N-Terminal Brain Natriuretic Peptide (NTproBNP). Echocardiograms allowed analysis of septal, left ventricular free wall and right ventricular free wall tissue velocities during systole and diastole. Before the RAAM cTnI levels were below the assay detection level (0.02 ng.ml -1). In three riders cTnI peaked on day one (0.03 ng.ml -1) and returned below detection levels post race. In the 4 th rider cTnI peaked on day 5 (0.08 ng.ml -1) and was still elevated post-race. Both CK-MB and H-FABP were increased during the RAAM in all 4 cyclists. In three riders H-FABP peaked on day one (3.49 to 5.09 ng.ml -1) and declined over the rest of the RAAM. In the final rider H-FABP peaked on day two (5.90 ng.ml -1) and then dropped back to baseline by the post-RAAM assessment. Interestingly, changes in H-FABP mirrored, temporally, changes in CK-MB in places and this may reflect an association with skeletal muscle damage. Data for GPBB value to (2.9 - 149.6 ng.ml -1) and NTproBNP value to (27.3 - 310.0 ng.L -1) were variable but again was elevated in all riders during the course of the RAAM. Changes in ventricular wall tissue velocities were minor and not cumulative. Peak atrial diastolic tissue velocity in the left ventricular free wall increased (P < 0.05) from 11 to 18 cm.s -1 over the last two race days but this did not significantly impact the ratio of early to late diastolic wall motion. Cardiac biomarkers were elevated during the completion of the RAAM in all 4 cyclist but changes were not cumulative which suggest that the hearts of the cyclists coped well with the extreme cardiac work demanded by this ultra-endurance exercise challenge.