Variability related to image acquisition and interpretation is an important issue of echocardiography in clinical trials. Nevertheless, there is no broadly accepted standard method for quality assessment of echocardiography in clinical research reports. We present analyses based on the echocardiography quality-assurance program of the ongoing STAAB cohort study (characteristics and course of heart failure stages A–B and determinants of progression). In 43 healthy individuals (mean age 50 ± 14 years; 18 females), duplicate echocardiography scans were acquired and mutually interpreted by one of three trained sonographers and an EACVI certified physician, respectively. Acquisition (AcV), interpretation (InV), and inter-observer variability (IOV; i.e., variability between the acquisition-interpretation sequences of two different observers), were determined for selected M-mode, B-mode, and Doppler parameters. We calculated Bland–Altman upper 95% limits of absolute differences, implying that 95% of measurement differences were smaller/equal to the given value: e.g. LV end-diastolic volume (mL): 25.0, 25.0, 27.9; septal e′ velocity (cm/s): 3.03, 1.25, 3.58. Further, 90, 85, and 80% upper limits of absolute differences were determined for the respective parameters. Both, acquisition and interpretation, independently and sizably contributed to IOV. As such, separate assessment of AcV and InV is likely to aid in echocardiography training and quality-assurance. Our results further suggest to routinely determine IOV in clinical trials as a comprehensive measure of imaging quality. The derived 95, 90, 85, and 80% upper limits of absolute differences are suggested as reproducibility targets of future studies, thus contributing to the international efforts of standardization in quality-assurance.