Collagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability. Copyright © 2012 Biophysical Society