Disabling generalized fatigue and muscle fatiguability are common features of post-poliomyelitis syndrome (PPS). In 17 fatigued PPS patients, we measured jitter on stimulation single-fiber electromyography (S-SFEMG) for at least 3.5 min before and after i.v. injection of 10 mg edrophonium. We observed reduction in jitter (defined as a significant difference in jitter means before and after edrophonium, unpaired t-test P < 0.05) in 7 patients, no change in 8, and a significant increase in 2 patients. Blinded to their edrophonium results, the 17 patients were treated with pyridostigmine 180 mg/day for 1 month, with a subjective improvement of fatigue in 9 patients, and with a significant reduction in mean Hare fatigue scores in the entire group of 17 patients (pre = 2.71, and post = 1.71; Wilcoxan signed rank sum test, P < 0.05). Edrophonium-induced reduction of jitter on S-SFEMG was significantly associated with pyridostigmine-induced subjective improvement of fatigue (Fisher's exact test, P < 0.04). A significant reduction in fatigue with pyridostigmine was observed only in the 7 patients who experienced a significant reduction in jitter with edrophonium (Wilcoxan signed rank sum test, P = 0.03). In addition, the 9 pyridostigmine responders experienced a significant reduction in jitter means pre- and post-edrophonium (100% vs. 88%, Bonferroni corrected, P < 0.01). We conclude that neuromuscular transmission as measured by jitter on S-SFEMG can improve with edrophonium in a proportion of PPS patients, and that generalized fatigue and muscle fatiguability in some patients with PPS may be due to anticholinesterase-responsive NMJ transmission defects.