Altered shoulder muscle activity is frequently believed to be a pathogenetic factor of subacromial impingement (SI) and therapeutic interventions have been directed towards restoring normal motor patterns. Still, there is a lack of scientific evidence regarding the changes in muscle activity in patients with SI. The aim of the study was to determine and compare the activity pattern of the shoulder muscles in subjects with and without SI. Twenty-one subjects with SI and 20 healthy controls were included. Electromyography (EMG) was assessed from eight shoulder muscles from both shoulders during motion. In the symptomatic shoulder, there was a significantly greater EMG activity during abduction in the supraspinatus and latissimus muscles and less activity in serratus anterior compared to the healthy subjects. During external rotation, there was significantly less activity of the infraspinatus and serratus anterior muscles on the symptomatic side compared to the healthy subjects. On the asymptomatic side, the groups showed different muscle activity during external rotation. Our findings of an altered shoulder muscle activity pattern on both the symptomatic and asymptomatic side in patients indicate that the different motor patterns might be a pathogenetic factor of SI, perhaps due to inappropriate neuromuscular strategies affecting both shoulders.