The knowledge of dynamic changes in the vascular system has become increasingly important in ensuring the safety and efficacy of endovascular devices. We developed new methods for quantifying in vivo three-dimensional (3D) arterial deformation due to pulsatile and nonpulsatile forces. A two-dimensional threshold segmentation technique combined with a level set method enabled calculation of the consistent centroid of the cross-sectional vessel lumen, whereas an optimal Fourier smoothing technique was developed to eliminate spurious irregularities of the centerline connecting the centroids. Longitudinal strain and novel metrics for axial twist and curvature change were utilized to characterize 3D deformations of the abdominal aorta, common iliac artery, and superficial femoral artery (SFA) due to musculoskeletal motion and deformations of the coronary artery due to cardiac pulsatile motion. These illustrative applications show the significance of each deformation metric, revealing significant longitudinal strain and axial twist in the SFA and coronary artery, and pronounced changes in vessel curvature in the coronary artery and in the inferior region of the SFA. The proposed methods may aid in designing preclinical tests aimed at replicating dynamic in vivo conditions in the arterial tree for the purpose of developing more durable endovascular devices including stents and stent grafts.