Abstract The aim of this study was to investigate how exercise influenced endogenous pain modulation in healthy controls, shoulder myalgia patients and fibromyalgia (FM) patients. Twenty-one healthy subjects, 20 shoulder myalgia patients and 20 FM patients, all females, participated. They performed standardized static contractions, that is, outward shoulder rotation (m. infraspinatus) and knee extension (m. quadriceps). Pressure pain thresholds (PPTs) were determined bilaterally at m. infraspinatus and m. quadriceps. During contractions PPTs were assessed at the contracting muscle, the resting homologous contralateral muscle and contralaterally at a distant site (m. infraspinatus during contraction of m. quadriceps and vice versa). Myalgia patients had lower PPTs compared to healthy controls at m. infraspinatus bilaterally ( p < 0.01), but not at m. quadriceps. FM patients had lower PPTs at all sites compared to healthy controls ( p < 0.001) and myalgia patients ( p < 0.001). During contraction of m. infraspinatus PPTs increased compared to baseline at the end of contraction in healthy controls (all sites: p < 0.003), but not in myalgia or FM patients. During contraction of m. quadriceps PPTs increased compared to baseline at the end of contraction in healthy controls (all sites: p < 0.001) and myalgia patients (all sites: p < 0.02), but not in FM patients. In conclusion, we found a normal activation of endogenous pain regulatory mechanisms in myalgia patients during contraction of the non-afflicted m. quadriceps, but a lack of pain inhibition during contraction of the painful m. infraspinatus. FM patients failed to activate their pain inhibitory mechanisms during all contractions.