Introduction: muscle pain/fibromyalgia (FM) is common among individuals with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We recently demonstrated that during orthostatic stress testing, adults with ME/CFS reported increased pain. In the current study, we hypothesized that pain pressure thresholds (PPT) would decrease and temporal summation (windup) would increase after head-up tilt testing (HUT), and that the presence of co-morbid FM would be associated with greater change in both measures. Methods and Results: 248 ME/CFS patients (164 with FM and84 without FM), and 22 healthy controls (HC) were analyzed. In HC there were no significant differences in PPT between pre- and post-HUT (finger: from 4.7(1.6) to 4.4(1.5); shoulder: from 2.8(1.0) to 2.9(1.0)). In ME/CFS patients with and without FM, a significant decrease in PPT post-HUT was found compared to HC (both p<0.0001). Patients with FM had a lower PPT pre- and post-HUT (finger: from 2.0(0.9) to 1.5(0.8); shoulder: from 1.2(0.5) to 1.0(0.5) compared to patients without FM (finger: from 5.0(1.6) to 3.3(1.5); shoulder: from 2.2(0.9) to1.9(1.0) (p ranging from 0.001 to <0.0001). In contrast to HC in ME/CFS patients windup was increased compared to HC pre-HUT (both p<0.0001), but did not significantly change post-HUT. Conclusions: Pressure pain threshold decreased in ME/CFS patients with or without fibromyalgia after head-up tilt test (HUT), compared to healthy controls. Windup pre-and post-HUT was significantly higher compared to healthy controls, but did not change from pre- to post-HUT. These results demonstrate that, like exercise, orthostatic stress can negatively influence the physiology of pain perception in ME/CFS.