Abstract: Long-term neck flexion posture is a common awkward posture resulting from long-term head-down work, long-term looking at a computer screen, and long-term playing with a mobile phone. Fatigue and chronic injury of cervical muscles are easily caused by long-term bowing of the head. Long-term bowing of the head to play with a mobile phone causes injury to the cervical spine. Long-term neck muscle contraction is an important cause of fatigue and chronic injury of the neck muscles and tissues. Therefore, it is of significance to analyze the changes of muscle activity during long-term neck muscle contraction and to determine the time threshold of neck muscle fatigue to reduce the damage caused by neck muscle fatigue. To quantitatively evaluate the effect of long-term head-down playing with a mobile phone on cervical spine fatigue, 20 healthy subjects were selected and kept the head-down angle between 40° and 60° for 3 h. On the basis of the analysis of cervical spine muscle architecture and anthropometry, the surface electromyography (sEMG) of the sternocleidomastoid, cervical gripper, and shoulder trapezius muscles was recorded. The original sEMG data were processed by filtering, rectifying, and amplitude standardization. The EMG values every 60 s were integrated, and their mean power frequency (MPF) was calculated. Results show that the fluctuation of the integral EMG is regular and the decrease after the initial increase indicates that the muscle is in the fatigue state. The MPF values of different muscles have obvious differences, which determine the duration of fatigue tolerance of the muscle. Moreover, the MPF does not exhibit a simple linear relationship during the entire bowing process. The results also show that negative MPF accumulation can be used to assess neck muscle fatigue. The sternocleidomastoid muscle is in the fatigue state in 20 min, whereas the cervical gripper and shoulder trapezius muscles are temporarily fatigued in approximately 20 min and in the final fatigue state in 75–100 min. Therefore, it is suggested that the duration of continuous bowing should not exceed 20 min.