This study aims to clarify some of the issues associated with the reliable measurement of muscle thickness on ultrasonographic images of the musculoskeletal system, namely the repeatability of measurements in different time frames, the effect of body side selection, and the effect of scan orientation. Ultrasound scans were performed on muscles associated with essential daily activities: geniohyoid, masseter, anterior arm muscles, rectus femoris, vastus intermedius, tibialis anterior, and gastrocnemius. Measurements of the muscle thickness were performed and repeated after 1, 6, and 24 h, on both dominant and nondominant side, using both transverse and longitudinal scans. Thirteen healthy volunteers (eight males and five females, mean age = 24 years, SD = 2.86, range = 19–29) were included. The intraclass correlation coefficient (ICC) was calculated between the baseline and the 1-, 6-, and 24-h interval, using a two-way mixed model of absolute agreement. The ICC ranged from 0.295 for the longitudinal scan of the left masseter muscle in the 6-h interval to 0.991 for the longitudinal scan of the nondominant anterior arm muscles in the 24-h interval. The results indicate that there is variable reliability of the measurements depending on the muscle, time frame, body side, and scan orientation. Consequently, the choice of these parameters can affect the validity of the measurements. Further investigation on a larger scale is required to establish the preferred parameters for each anatomical site.