The clinical utility of regional strain measurements in echocardiography is challenged by suboptimal reproducibility. In this study, we aimed to evaluate the test-retest reproducibility of regional longitudinal strain (RLS) per coronary artery perfusion territory (RLSTerritory) and basal-to-apical level of the left ventricle (RLSLevel), measured by a novel fully automated deep learning (DL) method based on point tracking. We measured strain in a dual-centre test-retest data set that included 40 controls and 40 patients with suspected non-ST elevation acute coronary syndrome. Two consecutive echocardiograms per subject were recorded by different operators. The reproducibility of RLSTerritory and RLSLevel measured by the DL method and by three experienced observers using semi-automatic software (2D Strain, EchoPAC, GE HealthCare) was evaluated as minimal detectable change (MDC). The DL method had MDC for RLSTerritory and RLSLevel ranging from 3.6 to 4.3%, corresponding to a 33-35% improved reproducibility compared with the inter- and intraobserver scenarios (MDC 5.5-6.4% and 4.9-5.4%). Furthermore, the DL method had a lower variance of test-retest differences for both RLSTerritory and RLSLevel compared with inter- and intraobserver scenarios (all P < 0.001). Bland-Altman analyses demonstrated superior reproducibility by the DL method for the whole range of strain values compared with the best observer scenarios. The feasibility of the DL method was 93% and measurement time was only 1 s per echocardiogram. The novel DL method provided fully automated measurements of RLS, with improved test-retest reproducibility compared with semi-automatic measurements by experienced observers. RLS measured by the DL method has the potential to advance patient care through a more detailed, more efficient, and less user-dependent clinical assessment of myocardial function.