Single fiber electromyography (SFEMG) is an advanced neurophysiological technique that is primarily used to assess the neuromuscular junction and disorders impairing adequate neuromuscular transmission. SFEMG measurements that are most useful clinically include jitter and blocking, which are quantitative and sensitive measures of neuromuscular transmission inefficiency; and abnormalities in these occur early in the course of diseases, often when standard electrodiagnostic studies are normal. Fiber density assesses the density of individual muscle fibers in each region of the muscle and is increased in a nonspecific manner in most neurogenic and myopathic disorders. SFEMG requires specialized recording equipment, which is used in the collection, display, analysis, reporting, and archiving of SFEMG data. Knowledge of technical limitations and potential pitfalls is vital in acquisition and analysis of SFEMG data. This chapter will review the concepts, methods, measurements, and technical factors related to single fiber EMG. SFEMG findings in various diseases will be reviewed.