BACKGROUND Altered recruitment of rotator cuff and scapulothoracic muscles has been identified in patients with subacromial impingement syndrome. To date, however, the cause-consequence relationship between pain and altered muscle recruitment has not been fully unraveled. METHODS The effect of experimental shoulder pain induced by injection of hypertonic saline in the supraspinatus on the activity of the supraspinatus, infraspinatus, subscapularis, trapezius, and serratus anterior activity was investigated during the performance of an elevation task by use of muscle functional magnetic resonance imaging in 25 healthy individuals. Measurements were taken at 4 levels (C6-C7, T2-T3, T3-T4, and T6-T7) at rest and after the elevation task performed without and with experimental shoulder pain. RESULTS During arm elevation, experimentally induced pain caused a significant activity reduction, expressed as reduction in T2 shift of the IS (P = .029). No significant changes in T2 shift values were found for the other rotator cuff muscles or the scapulothoracic muscles. CONCLUSIONS This study demonstrates that acute experimental shoulder pain has an inhibitory effect on the activity of the IS during arm elevation. Acute experimental shoulder pain did not seem to influence the scapulothoracic muscle activity significantly. The findings suggest that rotator cuff muscle function (infraspinatus) should be a consideration in the early management of patients with shoulder pain.