Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by intractable fatigue, postexertional malaise, and orthostatic intolerance, but its pathophysiology is poorly understood. Pharmacologic cholinergic stimulation was used to test the hypothesis that neurovascular dysregulation underlies exercise intolerance in ME/CFS.Does neurovascular dysregulation contribute to exercise intolerance in ME/CFS, and can its treatment improve exercise capacity?Forty-five subjects with ME/CFS were enrolled in a single-center, randomized, double-blind, placebo-controlled trial. Subjects were assigned in a 1:1 ratio to receive a 60-mg dose of oral pyridostigmine or placebo after an invasive cardiopulmonary exercise test (iCPET). A second iCPET was performed 50 min later. The primary end point was the difference in peak exercise oxygen uptake (VoTwenty-three subjects were assigned to receive pyridostigmine and 22 to receive placebo. The peak VoPyridostigmine improves peak VoClinicalTrials.gov; No.: NCT03674541; URL: www.gov.