Currently, no real-time three-dimensional echocardiographic (RT3DE) indices are recommended by the official guidelines for the assessment of diastolic dysfunction (DD). We hypothesized that recent developments in RT3DE imaging technology that allow dynamic quantification of both left ventricular (LV) volume and 3D myocardial deformation, could be utilized to objectively assess DD. Transthoracic RT3DE datasets were acquired (Philips iE33, X5 transducer, frame rate 19 ± 4) in 76 subjects, including 20 normal controls (NL), 16 mild DD, 20 moderate DD and 20 severe DD (grade 1, 2 and 3, respectively, using ASE guideline). Images were analyzed using prototype software (TomTec) that performs 3D speckle tracking to generate time curves of LV volume and segmental myocardial strain. Indices of diastolic LV function were calculated: volume at 25, 50 and 75 % of filling duration (FD) in percent of end-diastolic volume (volume index, LVVi), and rapid filling volume (RFV) fraction. Temporal indices included: FD in % of RR, and rapid filling duration (RFD) in % of FD. Additionally, longitudinal, radial and circumferential strains at 25, 50 and 75 % of FD were calculated. Inter-groups differences were tested using ANOVA. LVVi and RFV fraction showed a biphasic pattern with the severity of DD characterized by an initial decrease (grade 1), a pseudo-normalization (grade 2), and then an increase above normal (grade 3). FD progressively decreased with severity of DD. RFD was significantly increased in all 3 groups compared to NL. After normalization by peak systolic values, all three strain components showed a linear pattern with the severity of DD, suggesting potential clinical usefulness. This is the first study to show that current RT3DE technology allows combined quantitative analysis of LV volume and 3D myocardial strain, which is sensitive enough to demonstrate differences in myocardial relaxation in patients with different degrees of DD.