[Purpose] The purposes of this study were: 1) to validate a commercial software program using an optical-flow algorithm to measure the velocity of muscle movement; and 2) to determine optimal image quality and the size and location of regions of interest. [Materials and Methods] First, a block of pork thigh muscle was pulled at 33 different constant velocities. Subsequently, an accelerometer, a high-velocity camera, and ultrasonography were used to obtain measurements, and an Echolizer software was used to determine ultrasound-based velocities. Finally, the impact of the location and size of the regions of interest and the brightness and contrast of the images was analyzed. [Results] The regression equation was expressed as y=1.150 × −0.071 with a determination coefficient of 0.996. The average absolute error of the software was 0.02 mm/s, and the average relative error was 0.20% of the actual velocity between 2.5 and 16.5 mm/s after the regression equation was applied to the measured data. The accuracy of measurement was reduced owing to the increased size of the regions of interest, which included poor image quality or a deeper zone. [Conclusion] Our method of measuring muscle velocity using a custom program showed high validity and reliability. It is necessary to use the regression equation in the program to improve accuracy. However, the validity of the method could be reduced if the regions of interest involve deep tissues or areas with poor visualization of the muscle bundles, or if the brightness and contrast of the image are set inaccurately.
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