As the number of new transgenic mouse models of human neuromuscular disease continues to increase, the development of sophisticated electrophysiologic techniques for assessing the peripheral nervous system in these models has become important. Neuromuscular junction (NMJ) dysfunction, in particular, is often not detectable by morphologic or other techniques. To enable sensitive testing of murine NMJ function, we developed and tested a method for stimulated single fiber electromyography (S‐SFEMG) in the gastrocnemius muscles of anesthetized mice. Jitter was assessed by measuring the mean consecutive latency difference (MCD) of single fiber responses to sciatic nerve stimulation at 2 HZ. Mean MCD values in normothermic mice were in the range of 6–8 μs for different strains, with no MCD values exceeding 25 μs. Reduced core temperature (to 29°–30°C) resulted in increased jitter, whereas intubation and mechanical ventilation of mice did not alter these values. Intraperitoneal and intravenous injection of vecuronium, however, resulted in progressively increased jitter followed by blocking in continuously monitored fibers. These observations validate the utility of S‐SFEMG in mice as an index of NMJ function under a variety of physiologic conditions, and suggest that a high safety factor for neuromuscular transmission exists at mouse NMJs. © 2001 John Wiley & Sons, Inc. Muscle Nerve 24: 941–945, 2001