Although the mechanical phenomena associated with preconditioning are well-established, the underlying mechanisms responsible for this behavior are still not fully understood. Using quantitative polarized light imaging, this study assessed whether preconditioning alters the collagen fiber alignment of ligament tissue, and determined whether changes in fiber organization are associated with the reduced force and stiffness observed during loading. Collagen fiber alignment maps of facet capsular ligaments (n = 8) were generated before and after 30 cycles of cyclic tensile loading, and alignment vectors were correlated between the maps to identify altered fiber organization. The change in peak force and tangent stiffness between the 1st and 30th cycle were determined from the force-displacement response, and the principal strain field of the capsular ligament after preconditioning was calculated from the fiber alignment images. The decreases in peak ligament force and tangent stiffness between the 1st and 30th cycles of preconditioning were significantly correlated (R ≥ 0.976, p < 0.0001) with the change in correlation of fiber alignment vectors between maps. Furthermore, the decrease in ligament force was correlated with a rotation of the average fiber direction toward the direction of loading (R = −0.730; p = 0.0396). Decreases in peak force during loading and changes in fiber alignment after loading were correlated (p ≤ 0.0157) with the average principal strain of the unloaded ligament after preconditioning. Through the use of a vector correlation algorithm, this study quantifies detectable changes to the internal microstructure of soft tissue produced by preconditioning and demonstrates that the reorganization of the capsular ligament’s collagen fiber network, in addition to the viscoelasticity of its components, contribute to how the mechanical properties of the tissue change during its preconditioning.