In this study, the interactive effects of pyridostigmine, a pretreatment drug against nerve agents, and exercise training on muscle tension were investigated in the mouse lower extremity anterior muscular compartment by dorsiflexion of the foot with stimulation of the peroneal nerve. Acetylcholinesterase (AChE), lipid peroxidation (in terms of the end-product malondialdehyde, MDA) and creatine phosphokinase (CPK) activity in the muscle were correlated with muscle tension. Male NIH Swiss mice were divided into four groups and treated as follows: (1) sedentary control; (2) pyridostigmine (1.2 mg/kg orally) daily for the 5th and 6th weeks; (3) exercise training for 10 weeks; and (4) pyridostigmine plus exercise training for 10 weeks. Experiments on muscle tension were conducted 4 weeks after the last dose of pyridostigmine or saline and 24 h after exercise. The muscle tension was measured in right and left legs using a tension transduction device connected to a polygraph. After muscle tension recording, mice were killed, blood and triceps muscle were isolated, and plasma CPK and muscle AChE activities, and MDA were determined. There was a significant increase in the muscle tension (P<0.05) in the group treated with pyridostigmine plus exercise as compared to the control and exercise groups. The pyridostigmine plus exercise group also showed a significant reduction in AChE activity (P<0.01) and enhanced MDA (P<0.05) in the triceps muscle. These results suggest that subchronic dosages of pyridostigmine and interaction with exercise training result in the delayed effects of reduction in muscle AChE activity and enhanced muscle tension.