Increased sensitivity to atracurium in the high-dose pyridostigmine groups at 14 and 28 days, with and without removal of pyridostigmine 24 h before neuromuscular assessment suggests decreased margin of safety. Incremental doses of atracurium were administered and dose–response curves at both 14 and 28 days of pyridostigmine administration were calculated for each group. The 95% CIs of the doses to produce a 50% neuromuscular paralysis are presented as horizontal bars. The doses differ significantly if the bars do not overlap. The animals receiving high-dose pyridostigmine showed a significant leftward shift in the dose–response curves at both 14 and 28 days of pyridostigmine infusion, indicating that the neuromuscular transmission was not as robust compared with controls, irrespective of whether or not the osmotic pump infusing pyridostigmine was removed 24 h before pharmacological testing of neuromuscular function. The 5 mg·kg⁻¹·day⁻¹ pyridostigmine dose did not impair neurotransmission.