--- - |2 Summary This in vivo study tested the hypothesis that the modulation of acetylcholine (ACh) release by the M1 muscarinic receptor (mAChR) in the neuromuscular junction of disused muscles may affect the tensions of the muscles during the neuromuscular monitoring of a rocuronium‐induced neuromuscular block and compared the results with those obtained from normal muscles. A total of 20 C57BL/6 (wild‐type) and 10 α7 knock out (α7KO) mice were used in this experiment. As a pre‐experimental procedure, knee and ankle joints of right hind limbs were fixed by needle pinning at the 90° flexed position. After 2 weeks, the main experiment was performed. Both tendons of the tibialis anterior (TA) muscles were obtained, and the muscle tensions were recorded while the dose‐responses of rocuronium were measured three times in the same mouse by the serial administration of pirenzepine (0, 0.001 and 0.01 μg/g). Weight losses were observed after 2 weeks of immobilization in both groups, and a decrease in the mass of TA muscles at the immobilized side was observed compared to those of the contralateral nonimmobilized side. Tension depression of the TA muscles at immobilized side of the α7KO group was faster than those of the wild‐type group, but these differences decreased after the administration of pirenzepine. The tension depressions were similar regardless of the pirenzepine doses at the same side in the group. Tension depression may become more rapid in the α7 AChR‐expressed disused muscles by the decreased release of ACh release upon neuronal firing by the blockade of facilitatory M1 mAChR - 'Clinical and Experimental Pharmacology and Physiology, Volume 45, Issue 12, Page 1309-1316, December 2018. '