Existing clinical tests for infraspinatus lack accuracy and differentiation from supraspinatus is difficult. We aimed to find a position that isolated infraspinatus contraction from supraspinatus and to analyse the contraction characteristics of lower (oblique) and upper (transverse) parts of infraspinatus.Within-participant, repeated measures experimental study.Intramuscular electromyography was used to measure the level of activation (electromyographic amplitude as a percentage of maximal voluntary contraction) of infraspinatus and supraspinatus on 15 healthy participants. Participants produced an isometric external rotation force at the shoulder, against manual resistance in shoulder positions of neutral, flexion, abduction and extension. Longitudinal force along the humeral axis was also applied.The two parts of infraspinatus demonstrated different patterns of electromyographic activation. The oblique part of infraspinatus was "markedly active" in all positions while the transverse part was mostly "moderately active". Comparing supraspinatus with infraspinatus, it was found that infraspinatus was significantly more active than supraspinatus from the positions of shoulder flexion and neutral with the highest ratios observed in the position of shoulder flexion. Longitudinal humeral force was not an important factor.If isometric external rotation of the shoulder is performed against resistance, the oblique part of infraspinatus will be working harder than the transverse part, irrespective of shoulder position. If differentiation of infraspinatus contraction from supraspinatus is desired, external rotation should be performed from a position of shoulder flexion or neutral. Resisted external rotation in shoulder flexion may form the basis of the development of a more accurate clinical test for infraspinatus.