BACKGROUND: Strengthening the infraspinatus is important in shoulder rehabilitation. Changes in infraspinatus activity with changing load and position have not been quantified. We sought to determine the most appropriate load and posture for early infraspinatus strengthening by assessing for changes in electromyographic activity in the healthy infraspinatus and other shoulder girdle muscles during isometric external rotational exercise under different loads with the shoulder adducted in the supine and seated positions. METHODS: Sixteen healthy adults (30 shoulders) performed isometric shoulder external rotation in the sitting and supine positions, starting with the shoulder and forearm in neutral position and the elbow flexed 90°. Loads (0.5 kg, 1 kg, and 2 kg) were applied at rest. We assessed the infraspinatus, upper trapezius, posterior deltoid, and biceps brachii. For analysis, we used the mean percentage of maximum voluntary muscle contraction (%MVC) value measured during each isometric contraction divided by the maximum voluntary muscle contraction (MVC) of each muscle. RESULTS: In the infraspinatus and posterior deltoid, significant interaction was observed between body position and load. Compared to the sitting position, an increase in activity in the supine position was attenuated as load increased, especially at 2 kg. The supine values of the upper trapezius and biceps brachii were always significantly lower than those in the sitting position regardless of load. CONCLUSION: The activity of the infraspinatus can be increased gradually during rehabilitation by beginning in the supine position, which assures low activity of the upper trapezius and biceps brachii. Exercise with the shoulder adducted in the supine position can strengthen the infraspinatus gradually and avoid compensatory mobility. LEVEL OF EVIDENCE: Level 3.