Our recent findings in athletes with chronic ankle instability (CAI) revealed increased tone and stiffness alongside reduced elasticity in the peroneus longus (PL) during myotonometric (MYO) measurements at rest, suggesting diagnostic relevance. MYO recordings during muscle contraction in healthy subjects showed an active muscle stiffness influence on MYO parameters, suggesting its potential impact on CAI-related MYO findings. However, it remains unknown whether PL stiffening observed recently in CAI athletes at rest can also be detected while PL muscle contraction. This study, using myotonometry, examines the PL mechanical properties during a motor task mimicking PL’s biomechanical function, i.e., simultaneous isometric foot pronation and plantar flexion (IFPPF) at 30 % and 100 % of maximal voluntary contraction (MVC) in athletes with CAI. Nineteen adult male athletes with CAI (per International Ankle Consortium criteria) and 19 control (CO) athletes without lateral ankle sprain incidents comprised the study groups. Both groups had similar anthropometric parameters and training volume. Simultaneous force and MYO measurements were performed at 30 % and 100 % of MVC-IFPPF, using a MyotonPRO® device. Five MYO parameters were recorded in the PL: frequency, stiffness, decrement, relaxation time, and creep. No significant inter-group differences were observed in MYO parameters and force values measured during the 30 % and 100 % of MVC-IFPPF. This study, employing myotonometry, is the first to demonstrate the lack of significant differences between CAI and CO athletes in the MYO parameters measured in the PL muscle at submaximal and maximal contraction during simultaneous IFPPF, contrasting with our previous MYO results in CAI at rest.