The optimal combination of region-of-interest (ROI) size and strain length (SL) allowing two-segment strain and strain rate analyses in term neonates was investigated. The impact of different ROI sizes and SLs on the strain and strain rate beat-to-beat variation (BBV) was assessed in 80 good-quality tissue velocity images. Both BBVs decreased with increased ROI length and with increased SL (p < 0.05). There were no significant differences in the BBVs for ROI width 2, 3 and 4 mm (p > 0.05). Among the combinations eligible for two segment analysis, the lowest BBVs were found using SL 10 mm, ROI length 1 mm and ROI width 3 mm. Using this combination, the mean difference between the single-cycle value and two-cycle compound value for peak systolic strain rate was 6.2%, peak systolic strain was 2.9% and end systolic strain was 3.2% of the two-cycle compound mean values. Hence, strain and strain rate measurement in tissue velocity images in neonates is feasible and reliable.