Recent studies on fixation of syndesmotic injuries associated with rotational type ankle fractures suggest that the clinical paradigm may be shifting towards dynamic fixation of the ankle syndesmosis. However, knowledge of dynamic properties of the syndesmosis in vivo is limited. The objective of this study is to evaluate the reliability of dynamic –in vivo– computed-tomography (CT) assessment of the ankle syndesmosis. Patients: Twelve subjects with a clinical history of ankle instability, but without suspected syndesmotic injury. Intervention: CT-scans of one ankle in neutral and four extreme positions (dorsiflexion with/without eversion, plantarflexion with/without inversion) using a CT-compatible loading platform. Custom axial slices were created from each scan. Main outcome measures: Previously validated CT-measurements were used to quantify the geometric dimensions of the syndesmosis in the various positions: length of the incisura (LI), fibular length (FL), sagittal translation of the fibula (ST), tibiofibular clear space (TFCS), anterior/posterior widths of the syndesmosis (AW, PW) and fibular orientation. Statistical analysis: Intra- and inter-observer reliability was calculated using intraclass correlation-coefficient (ICC). Additionally, smallest detectable change (SDC; minimal displacement that ensures displacement is not result of measurement error) was calculated. Results: All measurements revealed excellent intra-observer ICC (range 0.89-0.98). Inter-observer ICCs were good to excellent (range 0.63-0.97). Upon ankle movement, CT- measurements showed significant displacement from neutral position to each extreme position (ΔTFCS, ΔST, ΔTFCS, ΔAW, ΔPW), all larger than their respective SDCs. Conclusions: Dynamic CT assessment and quantification of motion in the horizontal plane of the ankle syndesmosis is a clinically reliable technique.