A prospective cohort study. This study aims to assess the influence of the number of fused segments on cervical paravertebral muscles by comparing the changes of the cervical flexion relaxation phenomenon (FRP) after single-level versus multi-level anterior cervical discectomy and fusion (ACDF). A total of 115 patients underwent ACDF were retrospectively recruited, and divided into 1-level group (n=44), 2-level group (n=40) and 3-4-level group (n=31). The flexion relaxation experiment was carried out 3 days preoperatively and 12 months postoperatively by surface electromyography (SEMG). Patients were examined by the neck Visual Analogue Scale (VAS), the cervical Japanese Orthopaedic Association (JOA) score, Neck Disability Index (NDI) and C2-7 range of motion (ROM). There was a significant difference in the time-related changes of FRR among the three groups before and after surgery (F=85.701, P<0.001). 35 patients (79.55%) with 1 level ACDF and 11 patients (27.5%) with 2 levels ACDF had FRP restored to normal at 12 months postoperatively. However, only one patient (3.33%) had normalized FRP after 3-4 levels ACDF. There were significant differences in the time-related changes of the normalized SEMG RMS values in each phase before and after surgery (P=0.018, P<0.001, P<0.001, P<0.001). A significant correlation was found between the changes in C2-7ROM and FRR in three groups (P=0.007, 0.003 and 0.036). Single-level ACDF contributes to normalize the FRP of cervical paravertebral muscles, which, however, is not ideally recovered by 2-level ACDF. On the contrary, 3 or 4-level ACDF could not normalize the cervical FRP. Our research supported the passive structure hypothesis.