Objective: The aim of this study was to investigate the inter-rater and intra-rater reliability of rehabilitative ultrasound imaging (RUSI) for measurement of muscle thickness with changes in angles of the gluteus maximus (GM) at rest and during contraction. Design: Cross-sectional study. Methods: Twenty-two healthy men volunteered for this study. GM muscle images were obtained in the resting position and during prone hip extension with knee flexion at hip abduction angles of 0° and 30°, respectively. Two examiners randomly measured the thickness of the GM twice in three different positions. The first position was a comfortable prone position. The second position was prone hip extension with knee flexion (PHEKF) to 90°. The third position was achieved by hanging a 1-kg weight on the ankle of the lifted leg during PHEKF with the angle of the lifted leg the same as the second position. Intra-class correlation coefficients (ICCs), standard error measurements, and minimal detectable changes were used to estimate reliability. Results: The intra-rater reliability ICCs (95% confidence interval) of the GM were >0.870, indicating good reliability. Inter-rater reliability ICCs ranged from 0.668 to 0.913. The reliability of measurements of muscle thickness at each position was similar to the reliability of the angle change. Differences in muscle thickness and ratios for each position with 0° and 30° of hip abduction were not statistically significant. Conclusions: In the present study, the intra-rater reliability of muscle thickness measurements of the GM was good, and the inter-rater reliability was moderate to good. Reliable RUSI measurements of wide and large muscles, such as the GM muscle at rest and during contraction, are feasible. Further investigation is required to establish the reproducibility of the protocols presented in this study.