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Background Ankle sprain is one of the most common musculoskeletal injuries in sports, at work, and at home. Subjects who suffer from this injury may develop ankle instability. Functional instability has been associated with a high rate of resprain and impaired neuromuscular control in patients with ankle instability. Objective Measurement of neural and muscular mechanosensitivity after ankle sprain injury and establishment of the relationship between these variables. Methods A cross-sectional case-control study was performed with a sample of 58 students from Alcalá de Henares University (21 males and 37 females, mean age ± SD = 21 ± 3.7 years). Subjects were divided into two groups: a case group (N = 29, subjects with unstable ankle) and a control group (N = 29, healthy subjects). The pressure pain threshold (PPT) of the tibialis anterior, peroneus longus, and peroneus brevis muscles and mechanosensitivity of the common peroneus and tibial nerves were evaluated in all subjects through a manual mechanical algometer. Results Neuromuscular PPTs showed significant differences (P  < 0.05) between both groups, such that, compared with the control group, the case group exhibited significantly lower PPT levels. In the case group, a strong positive correlation was observed between neural and muscular homolateral mechanosensitivity in both lower limbs. Conclusions Participants with chronic ankle instability showed higher neuromuscular mechanosensitivity in muscles and nerves surrounding the ankle joint than healthy subjects. These findings indicate that low PPT values may be associated with symptoms that characterize this disease. [ABSTRACT FROM AUTHOR]