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Cross-over observational study.To determine the effect of exercise on mechanical ankle laxity, and how this effect differs between participants with chronic ankle instability (CAI) and healthy controls.Exercise increases knee joint laxity and increased laxity is thought to be a risk factor for ACL injury. Previous research at the ankle has examined whether tape or brace affects ankle laxity after exercise but not how exercise alone affects laxity at different time intervals.Twenty six female participants were recruited (22.9±3.1 years). Participants with CAI (n=13) reported ≥1 ankle sprain and a Cumberland Ankle Instability Tool (Youth) score ≤27. Control participants (n=13) had no ankle sprain history. Participants completed two test sessions on separate days in random order: an exercise (3 × 10 min structured aerobic exercise) and a rest session. Ligament laxity was measured at four time-points (baseline, 10 min, 20 min, 30 min) using an instrumented ankle arthrometer (Blue Bay Research), in the anterior-posterior (AP) and inversion-eversion (IE) directions. Repeated measure ANOVAs for AP and IE laxity were performed to compare a) exercise and rest and b) between groups.Increased AP displacement (1.55±2.01 mm, p=0.001) and IE rotation (1.32±2.22°, p=0.005) was observed during the first 10 min of exercise, which then plateaued. There was no difference between groups in either direction.Increased mechanical ankle laxity after exercise is consistent with knee research, however, results were within the standard error of measurement (SEM) of the arthrometer. Despite similar increase in mechanical ankle laxity with exercise in both groups, pre-existing increased mechanical instability in CAI participants might present a possible link to increased ankle injury risk and support the use of tape and brace to reduce ankle sprain rate in this population.