Individuals with chronic ankle instability (CAI) present with alterations in the compositional structure of their talar articular cartilage. These alterations likely influence how the talar cartilage responds to the loading associated with activities of daily living, such as walking. Ultrasonography has emerged as an alternative imaging modality for assessing the amount of cartilage deformation in response to loading because it is clinically accessible and cost effective for routine measurements. To (1) compare talar-cartilage deformation in response to a standardized exercise protocol between those with and those without CAI and (2) examine the association between spatiotemporal walking gait parameters and cartilage deformation. Case-control study. Research laboratory. A volunteer sample of 24 participants with self-reported CAI (age = 23.2 ± 3.9 years, body mass index [BMI] = 25.1 ± 3.7 kg/m2) and 24 uninjured controls (age = 24.3 ± 2.9 years, BMI = 22.9 ± 2.8 kg/m2). Spatiotemporal walking gait was first assessed from 5 self-selected trials using an electronic walkway with data sampled at 120 Hz. An 8- to 13-MHz linear-array ultrasound transducer placed transversely in line with the medial and lateral malleoli captured 3 images before and after a standardized loading protocol consisting of 30 single- and double-limb squats, 2-minute single-limb balance, and 10 single-legged drops from a 40-cm-height box. After controlling for BMI, we found that the participants with CAI had greater deformation than the uninjured control participants (P =.034). No other between-groups differences were observed (P values >.05). No significant partial correlations were noted between talar-cartilage deformation and spatiotemporal gait parameters when controlling for BMI (P >.05). Individuals with CAI had greater talar-cartilage deformation in response to a standardized exercise protocol than control individuals. The amount of talar-cartilage deformation was not associated with the spatiotemporal walking gait. [ABSTRACT FROM AUTHOR]