Injury to the shoulder represents a significant disability to the athlete participating in throwing and other overhead sporting activities. There are few scientifically based rehabilitation protocols regarding the most efficient exercises for the rotator cuff musculature. There is contradiction concerning the most efficient position for supraspinatus muscle electromyographic (EMG) activity. Because the supraspinatus is the most frequently injured musculotendinous structure of the shoulder, the most efficient position for testing and rehabilitation of this structure should be identified. The purposes of this study were to compare two test positions for supraspinatus EMG activity and maximal voluntary isometric contraction (MVIC) force development of the shoulder. Twenty-two subjects performed MVIC in the two recommended testing positions. A paired t-test revealed the prone position produced significantly greater (P = 0.04) EMG activity than the standing test position. An additional paired t-test revealed the standing position produced significantly greater (P = 0.0001) force than the prone position. Since the supraspinatus EMG activity was lowest in the position that produced the greatest shoulder MVIC force, we hypothesize that muscle substitution occurred. We recommend the prone position for supraspinatus rehabilitation and testing.