Although previous studies suggest that prolonged intense exercise such as marathon running transitorily alters cardiac function, there is little information regarding ultramarathon races. Aim of this study was to investigate the acute impact of ultra-endurance exercise (UEE) on heart, applying advanced strain imaging. Echocardiographic assessment was performed the day before and at the finish line of “Spartathlon”: A 246 Km ultra-marathon running race. 2D speckle-tracking echocardiography was performed in all 4 chambers, evaluating longitudinal strain (LS) for both ventricles and atria. Peak strain values and temporal parameters adjusted for heart rate were extracted from the derived curves. Out of 60 participants initially screened, 27 athletes (19 male, age 45±7 years) finished the race in 33:34:27(28:50:38-35:07:07) hours. Absolute values of right (RV) and left ventricular (LV) LS (RVLS -22.9±3.6 pre- to -21.2±3.0% post-, p=0.04 and LVLS -20.9±2.3 pre- to -18.8±2.0 post-, p=0.009) slightly decreased post-race, whereas atrial strain did not change. RV and LV LS decrease was caused mainly by strain impairment of basal regions with apical preservation. Inter-chamber relationships assessed through RV/LV, LV/LA, RV/RA and RA/LA peak values’ ratios remained unchanged from pre to post-race. Finally, UEE caused an extension of the systolic phase of cardiac cycle with concomitant diastole reduction (p<0.001 for all strain curves). Conclusively, ventricular LS strain as well as effective diastolic period slightly decreased, whereas atrial strain and inter-chamber relationships remained unchanged after running a 246-km-ultra-marathon race. These changes may be attributed to concomitant pre- and afterload alterations following UEE.