Previous attempts have been made to superimpose ultrasound images (overlapping images method). The merit of this method is that it enables capturing the entire morphology of the muscle and tendon in the longitudinal direction beyond the width of the ultrasound probe. However, the division points between tissues are identified visually, which could affect the reliability of the measurements. This study identified the division points for the overlapping images method using the following two procedures: (1) visual-only identification and (2) a combination of visual and custom-built software-based identification. Finally, the reliability of the measurements for each procedure was examined by estimating the intra- and inter-rater correlation coefficients (ICC 1.1, ICC 2.1). The lengths of the Achilles tendon, muscle-tendon unit, and gastrocnemius muscle were measured using the overlapping images method for 19 volunteers. The ICC 1.1 and ICC 2.1 scores for visual identification alone ranged between 0.61-0.91 and 0.70-0.93, respectively. The scores for the combination of visual and software-based identification ranged 0.90-0.99 and 0.58-0.96, respectively. The high intra- and inter-rater reliability in the overlapping images method was demonstrated in almost all measurements of the three tissue lengths and the applicability of overlapping images method was demonstrated. In particular, the intra-rater reliability was better when a combination of visual and custom-built software-based identification was used.
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