This prospective cohort study aimed to assess the influence of the number of fused segments in cervical paravertebral muscles by comparing the changes of the cervical flexion relaxation phenomenon (FRP) after single-level versus multilevel anterior cervical discectomy and fusion (ACDF). A total of 115 patients who had undergone ACDF were retrospectively recruited and divided into a 1-level group (n= 44), a 2-level group (n= 40) and a 3- to 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 using the neck visual analog scale, cervical Japanese Orthopedic Association score, Neck Disability Index, and C2-C7 range of motion (ROM). There was a significant difference in the time-related changes in flexion relaxation ratio (FRR) among the 3 study groups before and after surgery (F= 85.701; P < .001). Thirty-five patients (79.55%) with 1-level ACDF and 11 patients (27.5%) with 2-level ACDF had FRP were restored to normal at 12 months postoperatively; however, only 1 patient (3.33%) had normalized FRP after 3- to 4-level ACDF. There were significant differences in the time-related changes of the normalized SEMG root mean square values in each phase before and after surgery (P= .018, <.001, <.001, and <.001). A significant correlation was found between the changes in C2-C7 ROM and FRR in the 3 study groups (P= .007 for 1 level, P= .003 for 2 levels, and P=.036 for 3-4 levels). Single-level ACDF contributes to normalizing the FRP of cervical paravertebral muscles, which is not ideally recovered by 2-level ACDF. In contrast, 3- or 4-level ACDF could not normalize the cervical FRP. Our research supports the passive structure hypothesis.