The mechanism of myofascial pain syndrome (MPS) is unclear. Previous reports pointed out that pathogenesis of either referred pain or local twitch response in trigger point of MPS is related to the nerve degeneration and disintegration of motor and sensory nerves in spinal cord. The purpose of the present study is to investigate the evidence of neuromuscular transmission disorders in trigger point muscles of patients with MPS by using stimulated single-fiber electromyography (SFEMG) examination. Twenty-three patients with neck MPS and 16 age-matched controls attended for the present study. The jitter or mean consecutive difference (MCD) was calculated as the mean of the absolute differences for 30 consecutive interpotential intervals between stimuli and single-fiber potentials in trapezius and levator scapulae muscles. The relationship between the MCD values and the disease duration was measured. The results showed significantly increased MCDs in trapezius and levator scapulae muscles in MPS patients. MCD values measured in the trigger point muscles were found to be positively correlated with the disease duration. The present study with axonal microstimulation and SFEMG demonstrates a prominent evidence of neuroaxonal degeneration and neuromuscular transmission disorders in the trigger point muscles of MPS patients. The mechanism of MPS is possibly implicated with the degeneration of motor neurons.