Our understanding of in vivo Achilles tendon (AT) conditioning is limited to two-dimensional ultrasound measures of longitudinal deformation of the whole tendon. This study investigated the regional three-dimensional (3D) deformation of the AT during conditioning. Eighteen ATs were scanned using 3D freehand ultrasound during 10 successive 25 s submaximal (50%) voluntary isometric plantarflexion contractions. Longitudinal strain was assessed for the whole AT, aponeurosis, and free AT and transverse strain was assessed for the proximal-, mid-, and distal-portions of the free AT. Longitudinal conditioning of the whole AT was primarily driven by creep response of the free AT and transverse conditioning was greatest for the mid-portion of the free AT. Whole and free AT longitudinal strain increased up to the third contraction and were accompanied by a corresponding reduction in free AT cross-sectional area (CSA) strain in proximal-, mid-, and distal-portions. No significant changes in aponeurosis strain or tendon volume were detected between contractions. These findings suggest that conditioning alters free AT shape, with increased tendon length attained at the expense of reduction in free AT CSA. Although AT experiences different amounts of strain in different regions, the number of contractions required to reach steady-state strain during conditioning is uniform throughout the tendon.