Objectives. We tested the feasibility of real-time 3D echocardiographic (RT3DE) imaging to measure left heart volumes at different gravity during parabolic flight and studied the effects of lower body negative pressure (LBNP) as a countermeasure. Background. Weightlessness-related changes in cardiac function have been previously studied during space flights using both 2D and 3D echocardiography. Several technical factors, such as inability to provide real-time analysis and the need for laborious endocardial definition have limited its usefulness. RT3DE imaging overcomes these limitations by acquiring real-time pyramidal datasets encompassing the entire ventricle. Methods. RT3DE datasets were obtained (Philips 7500, X3) during breath-hold in 16 unmedicated normal subjects in upright standing position at different gravity phases during parabolic flight (normogravity, 1Gz; hypergravity, 1.8Gz; microgravity, 0Gz), with LBNP applied (-50 mmHg) at 0Gz in selected parabolas. Results. RT3DE imaging during parabolic flight was feasible in 14/16 subjects. Data were analyzed (Tomtec) to quantify left ventricular (LV) and atrial (LA) volumes at end-diastole and end-systole, which significantly decreased at 1.8Gz and increased at 0Gz. While ejection fraction did not change with gravity, stroke volume was reduced by 16% at 1.8Gz and increased by 20% at 0Gz, but was not significantly different from 1Gz values with LBNP. Conclusions. RT3DE during parabolic flight is feasible and provides the basis for accurate quantification of LV and LA volume changes with gravity. As LBNP counteracted the increase of LV and LA volumes caused by changes in venous return, it may be effectively used for preventing cardiac dilatation during microgravity.