Objective To study pressure pain threshold (PPT) of the spine and lower limbs for patients with acute unilateral neck pain and neck mobility deficits, and its relationship with cervical pain and cervical range of motion (ROM), so as to establish accurate quantitative assessment index by comparison with healthy controls. Methods A total of 48 patients with unilateral neck pain and neck mobility deficits (experimental group), as well as 48 healthy adults (control group) were included. The PPTs of the splenius capitis, upper fibers of trapezius, rhomboideus major, quadratus lumborum, midpoint of hamstring, popliteal fossa, mid and lower point of gastrocnemius, and plantar fascia at base of the second metatarsal were tested by using a handheld thresholds algometer. The pain score and cervical ROM were recorded. ResultsThere were significant differences in neck extension as well as rotation and lateral flexion of the affected side between experimental group and control group. The PPTs of splenius capitis, trapezius, mid and lower part of gastrocnemius in the affected side in experimental group were significantly different from those in the healthy side and in control group (P＜0.05). There was a negative correlation between neck pain and ROMs of neck extension, lateral flexion of the affected side and rotation of the affected side (P＜0.05). A correlation between neck pain and the PPTs of splenius capitis, upper trapezius, midpoint of gastrocnemius muscle and lower part of gastrocnemius muscles was also found (P＜0.05). Conclusions The PPTs of trapezius muscle, splenius capitis and gastrocnemius decrease in patients with acute neck pain and unilateral mobility deficits, and they are significantly correlated with neck pain and mobility deficits. This study provides references for quantitative assessment of patients with neck pain in clinic.