Abstract High failure rates of femoropopliteal artery reconstruction are commonly attributed to complex 3D arterial deformations that occur with limb movement. The purpose of this study was to develop a method for accurate assessment of these deformations. Custom-made stainless-steel markers were deployed into 5 in situ cadaveric femoropopliteal arteries using fluoroscopy. Thin-section CT images were acquired with each limb in the straight and acutely bent states. Image segmentation and 3D reconstruction allowed comparison of the relative locations of each intra-arterial marker position for determination of the artery’s bending, torsion and axial compression. After imaging, each artery was excised for histological analysis using Verhoeff–Van Gieson staining. Femoropopliteal arteries deformed non-uniformly with highly localized deformations in the proximal superficial femoral artery, and between the adductor hiatus and distal popliteal artery. The largest bending (11±3–6±1mm radius of curvature), twisting (28±9–77±27°/cm) and axial compression (19±10–30±8%) were registered at the adductor hiatus and the below knee popliteal artery. These deformations were 3.7, 19 and 2.5 fold more severe than values currently reported in the literature. Histology demonstrated a distinct sub-adventitial layer of longitudinally oriented elastin fibers with intimal thickening in the segments with the largest deformations. This endovascular intra-arterial marker technique can quantify the non-uniform 3D deformations of the femoropopliteal artery during knee flexion without disturbing surrounding structures. We demonstrate that 3D arterial bending, torsion and compression in the flexed lower limb are highly localized and are substantially more severe than previously reported.