Muscle thickness may now be measured in the clinic by ultrasonography. The purpose of this study was to test (i) whether the position of the transducer on the muscle affects muscle thickness measurements and (ii) whether measurements from identical locations vary over time. Muscle thickness of one masseter muscle side was measured in each of 30 volunteers. Measurements were recorded at five different locations on the lateral surface of each muscle. Four measurement sessions were performed, and two sets of measurements were recorded at each session. Spatial position of the transducer had a clear effect on the muscle thickness measurements (P < 0.0001). Measurements made at the most upper and middle level were less and greater (P < 0.0001), respectively, than those made at the upper, lower, and most lower levels. Measurements from various positions did not differ (P > 0.05) when made at the same level. Masseter thickness measurements recorded at a given site were consistent across all of the testing sessions (P=0.575). The data suggest that reliable interpretation of longitudinal measurements of masseter thickness obtained with ultrasonography requires accurate repositioning of the transducer. Ultrasonography may be used to investigate the effect of specific treatment modalities on masseter thickness measurements.