The prevalence of chronic trapezius myalgia is high in women with high exposure to awkward working positions, repetitive movements and movements with high precision demands. The mechanisms behind chronic trapezius myalgia are not fully understood. The purpose of this study was to explore the differences in protein content between healthy and myalgic trapezius muscle using proteomics. Muscle biopsies from 12 female cleaners with work-related trapezius myalgia and 12 pain free female cleaners were obtained from the descending part of the trapezius. Proteins were separated with two-dimensional differential gel electrophoresis (2D-DIGE) and selected proteins were identified with mass spectrometry. In order to discriminate the two groups, quantified proteins were fitted to a multivariate analysis: partial least square discriminate analysis. The model separated 28 unique proteins which were related to glycolysis, the tricaboxylic acid cycle, to the contractile apparatus, the cytoskeleton and to acute response proteins. The results suggest altered metabolism, a higher abundance of proteins related to inflammation in myalgic cleaners compared to healthy, and a possible alteration of the contractile apparatus. This explorative proteomic screening of proteins related to chronic pain in the trapezius muscle provides new important aspects of the pathophysiology behind chronic trapezius myalgia.