Botulinum toxin injection is an effective treatment for hemifacial spasm by causing pre-synaptic block at the neuromuscular junction. Its effects are temporary and repeated injections are required to maintain benefi t. Mild, permanent facial weakness is often thought to be due to prolonged nerve compression. To investigate the possibility of a chronic and persistent effect of botulinum toxin on neuromuscular transmission, we carried out stimulated single-fi bre electromyography to compare jitter of the affected orbicularis oculi in previously treated and treatment naïve hemifacial spasm patients. Previously treated patients were studied when the acute effects of the last botulinum toxin injection had worn off. We found mean jitter was signifi cantly higher in previously treated patients. Although, treated patients had longer duration of hemifacial spasm, mean jitter was dependent only on the number of previous botulinum injections and independent of the duration of hemifacial spasm and time from the last injection. This suggests a persistent and cumulative effect of botulinum toxin on neuromuscular transmission in patients treated for hemifacial spasm.