AbstractObjectiveThere is growing interest in speckle tracking echocardiography‐derived strain as a measure of left ventricular function in neonates. However, knowledge gaps remain regarding the effect of image acquisition and processing parameters on circumferential strain measurements. The aim of this study was to evaluate the effect of using different region of interest (ROI) widths on speckle tracking derived circumferential strain in healthy neonates.MethodsThirty healthy‐term‐born neonates were examined with speckle‐tracking echocardiography in the short‐axis view. Circumferential strain values were acquired and compared using two different ROI widths. Furthermore, strain values in the different vendor‐defined wall layers were also compared.ResultsIncreasing ROI width led to a decrease in global circumferential strain (GCS) in the midwall and epicardial layers, the respective decreases in strain being ‐23.4 ± .6% to ‐22.0 ± 1.1%, p < .0001 and 18.5 ± 1.7% to ‐15.6 ± 2.0%, p < .0001. Segmental analyses were consistent with these results, apart from two segments in the midwall. There was no statistically significant effect on strain for the endocardial layer. A gradient was seen where strain increased from the epicardial to endocardial layers.ConclusionIncreasing ROI width led to a decrease in GCS in the midwall and epicardium. There is an increase in circumferential strain when moving from the epicardial toward the endocardial layer. Clinicians wishing to implement circumferential strain into their practice should consider ROI width variation as a potential confounder in their measurements.