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Speckle tracking analysis (STE) of the left ventricle offers a new method to assess left ventricular (LV) diastolic function. LV diastolic strain measurements offer a non-invasive, global and direct measure of LV diastolic function. However, there is little data on normal values and the influence of anthropomorphic factors which is crucial in clinical practice for new techniques. The aims of this study were to formulate reference values for LV diastolic strain rate, elucidate effects of age and sex on LV diastolic strain analysis and compare STE measurements with conventional LV diastolic measurements. One-hundred-forty-seven healthy subjects aged 20–72 years (≥ 28 subjects per age decade) were prospectively included (Mean age 44 ± 13.7 years, 50% female) and examined with electrocardiography and 2D-echocardiography, including speckle tracking. Left ventricular peak early diastolic strain rate (Sre) was measured in the apical windows, using STE. Men had significantly lower LV Sre values than women (1.02 ± 0.22 vs. 1.18 ± 0.23, p value < 0.001). Left ventricular Sre was inversely associated with age, with values decreasing with ageing. An inverse relation was also found with blood pressure and body surface area. Linear regression analysis showed that LV Sre was independently associated with both age and sex. A multivariable linear regression analysis for LV Sre with conventional LV diastolic variables accounted for 70.9% of the variation of LV Sre, showing good model performance. Reference values for LV Sre are reported and found to be both age- and sex-dependent. Therefore we recommend age- and sex-specific references values to be used in daily clinical practice.