A marked reduction in upright stroke volume (SV) contributes substantially to orthostatic intolerance after exposure to spaceflight or bed rest. It is unclear whether slowed left ventricular (LV) relaxation and diastolic suction contribute to the reduction in SV or whether these changes are influenced by exercise training while in bed. Twenty-seven healthy adults completed 5 weeks of -6 deg head-down bed rest (HDBR). During HDBR, nine subjects were sedentary (NOEX), while 18 performed near-daily rowing ergometry (EX). Left ventricular mass, SV, LV end-diastolic volume (LVEDV), pulmonary capillary wedge pressure and Doppler ultrasound indices of LV function were collected pre- and post-HDBR during supine rest (twice) and during reduced LV loading (lower body negative pressure; LBNP) and increased LV loading (saline infusion). Post-HDBR, LV mass increased in the EX group, but decreased in the NOEX group. The reduction in SV and LVEDV during supine rest and LBNP were greater with NOEX in comparison to EX after HDBR. Peak early mitral annular velocity, isovolumic relaxation time, early propagation velocity, a non-invasive index of early diastolic filling and ventricular diastolic suction, and peak global longitudinal early strain rate were slowed during supine rest after HDBR with NOEX; however, these variables were either unaltered or the reduction was less prominent with EX. Doppler ultrasound measures of early diastolic filling, ventricular relaxation and diastolic suction were not significantly affected during LV unloading by LBNP after HDBR in either group. All Doppler indices were restored to pre-HDBR levels in both groups during saline infusion to normalize LV filling pressure after HDBR. It is concluded that Doppler indices of dynamic LV filling were reduced in both groups after HDBR; however, these effects were more pronounced in the NOEX group. Irrespective of group, post-HDBR Doppler parameters were restored when LV filling pressure was increased to pre-HDBR levels during saline infusion. Therefore, the reduction in upright SV after HDBR is more influenced by changes in LV loading conditions, namely left atrial pressure in the setting of LV remodelling, rather than ventricular relaxation and diastolic suction.