We retrospectively assessed the rest-phase images of 300 contrast stress- echocardiograms, during which very-low mechanical index (VLMI) imaging with ultrasound enhancing agents (UEAs) was routinely acquired in addition to greyscale echocardiography; intra- and inter-reader variability for left ventricle (LV) volumes and ejection fraction (LVEF) at rest was established in the overall cohort and normal values were calculated in the subset of subjects with no cardiac risk factors or cardiac disease and a normal stress-echocardiogram. Current recommendations for chamber quantification using echocardiography advise the use of UEAs in case of insufficient quality of endocardial visualization, but normal values for LV volumes and LVEF using UEAs have never been published using currently recommended VLMI method. Single-centre retrospective study, enrolling subjects referred to contrast stress- echocardiography for suspect coronary artery disease, including the acquisition of both standard 2D greyscale and bolus UEAs with VLMI, regardless of the image quality. This enables offline measurement of the LV volumes and LVEF for both greyscale and UEAs–VLMI images at rest in all subjects. Images were allocated to 3 readers in random order. Normal range for LV volumes and LVEF was also derived in a subset of apparently healthy subjects. In the 298 exams finally assessed, measurement variability among the three readers was lower with UEAs-VLMI, in particular for end-systolic volumes (intra-class correlation coefficient for concordance improved from 0.855 to 0.916, for LVEF from 0.68 to 0.783, p < 0.01), intra- observer variability reduced (Lin’s correlation coefficient for LVEF from 0.747 to 0.857, p < 0.01). Normal mean indexed LV end-diastolic volume with UEAs-VLMI, compared to greyscale imaging, was + 14 ml/m², while LVEF was + 5 to + 6% points. This is the largest study specifically addressing UEAs–VLMI imaging and comparing data with standard greyscale imaging; it demonstrates a reduction in measurement variability of LV volumes and LVEF. Normal reference values for VLMI ultrasound are reported for the first time in comparison with greyscale values.