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This study sought to find the most robust method for left ventricular (LV) rotation measurement by speckle tracking echocardiography (STE) with the new QLAB Advanced Quantification Software (version 6.0, Philips, Best, The Netherlands).The study population consisted of 40 non-selected patients (mean age 48 ± 18 year, 20 men) and 50 non-selected healthy volunteers (mean age 34 ± 12 year, 21 men). Feasibility and intra-observer reproducibility of the measurement of LV rotation parameters by STE were assessed for two different methods (Method A: six tracking points placed mid-myocardial and Method B: six tracking points placed endocardial and epicardial forming six myocardial segments). Subsequently, inter-observer and temporal reproducibility of the most robust method were assessed. Complete LV rotation assessment was more feasible with Method A (60 out of 90 subjects, 67% vs. 50 out of 90 subjects, 56%). In the 49 subjects in whom both Methods A and B were feasible, intra-observer reproducibility of LV rotation parameters was better with Method A (variabilities 2 ± 3 to 10 ± 9% vs. 2 ± 4 to 21 ± 18%). With this method, inter-observer variability varied from 4 ± 4 to 13 ± 9% and temporal variability from 4 ± 6 to 19 ± 15%.The most robust method to assess LV rotation with QLAB software is from the mid-myocardium. This method is feasible in approximately two-thirds of subjects and has good intra-observer, inter-observer, and temporal reproducibility, allowing to study changes over time in LV rotation in an individual patient.