An effect of aging on cardiac morphology and function has been shown in humans. In horses, cardiac wall motion analysis using two-dimensional speckle tracking (2D-ST) has not yet been reported. Our study included 57 horses of different warmblood breeds between 3 and 30 years old. Age had a significant influence on left ventricular free wall (LVFW) systolic strain rate (p ≤ 0.05) and early diastolic relaxation (p ≤ 0.01). In the interventricular septum (IVS), systolic (p ≤ 0.01) and late diastolic (p ≤ 0.05) contraction velocities also increased with age. In our study, 2D-ST revealed important information on myocardial function, which was most evident in the LVFW, where measurements were highly reproducible. Aging seems to be associated with structural changes within the myocardium and with decreasing contraction capacity in old animals. These physiological, age-related processes should be considered when performing cardiac wall motion analysis of the 2D-ST results for the LVFW and IVS in horses.