The contractile function of skeletal muscles is primarily regulated by the expression of myosin heavy chain (MHC) isoforms. Adult human skeletal muscles express three MHC isoforms (MHC-I, MHC-IIa and MHC-IIx). The muscles mainly expressing MHC-I are slow but resistant to fatigue, while those with major expression of MHC-IIa and MHC-IIx are fast and powerful but less resistant to fatigue. In this study, mRNA levels of the MHC isoforms were assessed in 24 human supraspinatus muscles by reverse-transcription polymerase chain reaction. The average expression of the MHC-I isoform was 36.72%, that of the MHC-IIa isoform was 33.52%, and the average expression of the MHC-IIx isoform was 29.76%. The higher average expression of the two MHC-II isoforms taken together (63.28%) indicates that the human supraspinatus muscle is a powerful, fast muscle with relatively low resistance to fatigue, in accordance with its role in the elevation of the upper extremity. In women, and more markedly in older women, the trend towards upregulation of the fast MHC-II isoforms and downregulation of the slow MHC-I isoform, which is absent in males, may improve our understanding of possible causes of the subacromial impingement syndrome.