Experimental studies implicate late systolic load as a determinant of impaired left-ventricular relaxation. We aimed to assess the relationship between the myocardial loading sequence and left-ventricular contraction and relaxation. Time-resolved central pressure and time-resolved left-ventricular geometry were measured with carotid tonometry and speckle-tracking echocardiography, respectively, for computation of time-resolved ejection-phase myocardial wall stress (EP-MWS) among 1214 middle-aged adults without manifest cardiovascular disease from the general population. Early diastolic annular velocity and systolic annular velocities were measured with tissue Doppler imaging, and segment-averaged longitudinal strain was measured with speckle-tracking echocardiography. After adjustment for age, sex, and potential confounders, late EP-MWS was negatively associated with early diastolic mitral annular velocity (standardized β=-0.25; P<0.0001) and mitral inflow propagation velocity (standardized β=-0.13; P=0.02). In contrast, early EP-MWS was positively associated with early diastolic mitral annular velocity (standardized β=0.18; P<0.0001) and mitral inflow propagation velocity (standardized β=0.22; P<0.0001). A higher late EP-MWS predicted a lower systolic mitral annular velocity (standardized β=-0.31; P<0.0001) and lesser myocardial longitudinal strain (standardized β=0.32; P<0.0001), whereas a higher early EP-MWS was associated with a higher systolic mitral annular velocity (standardized β=0.16; P=0.002) and greater longitudinal strain (standardized β=-0.24; P=0.002). The loading sequence remained independently associated with early diastolic mitral annular velocity after adjustment for systolic mitral annular velocity or systolic longitudinal strain. In the context of available experimental data, our findings support the role of the myocardial loading sequence as a determinant of left-ventricular systolic and diastolic function. A loading sequence characterized by prominent late systolic wall stress was associated with lower longitudinal systolic function and diastolic relaxation.