Epidemiological research provides strong evidence for a link between repetitive work (RW) and the development of chronic trapezius myalgia (TM). The aims were to further elucidate if an accumulation of sensitising substances or impaired oxygenation is evident in painful muscles during RW. Females with TM (n = 14) were studied during rest, 30 minutes RW and 60 minutes recovery. Microdialysate samples were obtained to determine changes in intramuscular microdialysate (IMMD) [glutamate], [PGE(2)], [lactate], and [pyruvate] (i.e., [concentration]) relative to work. Muscle oxygenation (%StO(2)) was assessed using near-infrared spectroscopy. During work, all investigated substances, except PGE(2), increased significantly: [glutamate] (54%, P < .0001), [lactate] (26%, P < .005), [pyruvate] (19%, P < .0001), while the %StO(2) decreased (P < .05). During recovery [PGE(2)] decreased (P < .005), [lactate] remained increased (P < .001), [pyruvate] increased progressively (P < .0001), and %StO(2) had returned to baseline. Changes in substance concentrations and oxygenation in response to work indicate normal increase in metabolism but no ongoing inflammation in subjects with TM.