Context: The supraspinatus is the most commonly affected muscle with rotator-cuff pathology and necessary for stability of the humeral head in the glenoid fossa. Rehabilitative ultrasound imaging (RUSI) of skeletal muscles provides a safe and clinically accessible measure of intact human muscle function at rest or during contracted states. The ability to perform accurate assessment of supraspinatus function has not been studied and may be of value in assessment and treatment. Objectives: To determine the validity and reliability of measures obtained using RUSI for assessing supraspinatus muscle at rest and contracted conditions. Design: Reliability and validity Setting: Outpatient physical therapy clinic. Subjects: 15 asymptomatic subjects age 30-49 y. Main Outcome Measures: The supraspinatus muscle was measured at rest and contracted with a 0.9-kg weight with the arm positioned in 45° of abduction in the plane of the scapula. Repeated ultrasound images of the supraspinatus were collected by 3 physical therapists on 2 separate days. Main Outcome Measures: Reliability was assessed with the intraclass correlation coefficient (ICC) and standard error of measurement (SEM). Validity was tested by comparing mean difference between active and passive states for all 3 rates on both days. Results: All ICC values were found to be at .9 or above. In addition, for all days and raters, the active condition was significantly thicker than the passive condition (P < .001). Conclusions: Thickness measures of the supraspinatus using RUSI, during passive and active conditions, demonstrate high interrater and intrarater reliability and can easily distinguish between active and passive states. These findings suggest that RUSI may provide an appropriate quantitative measure for changes in the thickness of supraspinatus that are important for determining improvement or deterioration in muscle function.