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Objectives: This study aimed to determine feasibility, reference intervals, and reproducibility of left ventricular ejection fraction (LVEF) and speckle‐tracking echocardiography (STE) in adult Syrian hamsters. Background: Syrian hamster is an experimental model for several heart diseases. Echocardiography allows the evaluation of structure and function with bidimensional conventional techniques and STE. However, there is no data regarding reference values for bidimensional LVEF and myocardial strain in hamsters. Methods: A total of 135 female Syrian hamsters were anesthetized and studied with a small animal dedicated echocardiography system. Echocardiography measurements were obtained from M‐mode and B‐mode images. Feasibility and 95% reference intervals were obtained for LVEF using three different approaches: LVEF_Teichholz (from M‐mode linear measurements), LVEF_BMode (from area‐length method), and LVEF_ STE (from strain), and for global longitudinal (GLS), circumferential (GCS), and radial (GRS) endocardial strain. Reproducibility was assessed as intra‐class correlation coefficients. Results: Feasibility of LVEF and endocardial strain was high (95% in FEVE_Teichholz, 93% in the LVEF_BMode, 84% in the LVEF_STE, 84% from PSLAX, and 80% from PSSAX). Values of LVEF_Teichholz were significantly higher than values of LVEF_BMode, and LVEF_STE‐derived methods (59.0 ± 5.8, 53.8 ± 4.7, 46.3 ± 5.7, p < 0.0001). The 95% reference intervals for GLS, GCS, and GRS were respectively −13.6(−7.5;‐20.4)%, −20.5 ± 3.1%, and + 34,7 ± 7.0%. Intra‐class correlation coefficients were 0.49 – 0.91 for LVEF measurements, 0.73 – 0.92 for STE, with better results for LVEF_Teichholz and GLS. Conclusions: Evaluation of LVEF by several methods and STE parameters is feasible in hamsters. Reference intervals for LVEF and STE obtained for this experimental animal model can be applied at future research. [ABSTRACT FROM AUTHOR]