Velocity vector imaging (VVI) software permits quantitative assessment of ventricular function through measurement of myocardial strain (S) and strain rate (SR). The purpose of this study was to define a reference range of ventricular S and SR values in normal adults using VVI software, and to describe the variability among observers and systems. Two-dimensional echocardiography was performed in 186 healthy adults free of cardiovascular disease or risk factors, followed by comprehensive ventricular S and SR analysis using VVI software. Images were acquired using three commercial ultrasound systems. The mean age of patients was 44 ± 16 years, and 114 (61 %) were female. Mean global left ventricular (LV) longitudinal, circumferential, and radial S and SR, and right ventricular (RV) longitudinal S and SR values are presented. Significant segmental variation in regional LV and RV S and SR was detected. Multivariate regression analysis demonstrated global longitudinal LV (p = 0.05) and RV (p = 0.002) S values decline significantly with age. The overall variability of S and SR values accounted for by patient demographic and hemodynamic variables was low (16 and 8 % for LV longitudinal S and SR, respectively). Interobserver agreement was very good, but was lowest for LV radial S and SR. There were no significant differences of LV and RV S and SR between ultrasound systems. Comprehensive reference values for the normal ranges of LV and RV S and SR measured using VVI software are presented. The ultrasound system used for image acquisition did not significantly influence results.