Extracellular matrix (ECM) proteins and their proteolytic enzymes, matrix metalloproteinases (MMPs), implicate in neuromuscular junctions (NMJs) function during development. However, their pathophysiological mechanisms in the diaphragm remain obscure, because a well-characterized ex vivo experimental system has still been lacking. In the study, we aim to develop a novel ex vivo method of sonomicrometry and evaluate validity of the method with a mouse diaphragm twitch after phrenic nerve injury. In an ex vivo experiment using phrenic nerve-injured mice, diaphragm twitch during electrical pulse stimulation of phrenic nerve was transiently suppressed on day 1. Recombinant MMPs administered in recording solution exerted dose-responsive suppression on the diaphragm twitch in normal mice tissue. Furthermore, gelatinolytic and immunoblot experiments were performed to evaluate MMPs' involvement and NMJs' insults. After nerve injury, (1) in vivo levels of MMPs were transiently upregulated at day 1 and (2) expressions of ECM proteins, agrin (nicotinic acetylcholine receptor stabilizer) and laminin, were transiently reduced at day 1 in the diaphragm. These alterations were cancelled by preinjection of the MMP inhibitor. In conclusion, MMPs hamper NMJ synaptic function in association with the impairment of ECM milieu. Our novel experimental method using ex vivo sonomicrometry is necessary for examining the molecular pathophysiolgy for the dysfunction of NMJs in the diaphragm. Synapse, 2012. © 2012 Wiley Periodicals, Inc.