Skin aging is a complex process that strongly affects the mechanical behavior of skin. This study aims at deciphering the relationship between age-related changes in dermis mechanical behavior and the underlying changes in dermis microstructure. To that end, we use multiphoton microscopy to monitor the reorganization of dermal collagen during mechanical traction assays in ex vivo skin from young and old mice. The simultaneous variations of a full set of mechanical and microstructural parameters are analyzed in the framework of a multiscale mechanical interpretation. They show consistent results for wild-type mice as well as for genetically-modified mice with modified collagen V synthesis. We mainly observe an increase of the tangent modulus and a lengthening of the heel region in old murine skin from all strains, which is attributed to two different origins that may act together: (i) increased cross-linking of collagen fibers and (ii) loss of water due to proteoglycans deterioration, which impedes inner sliding within these fibers. In contrast, the microstructure reorganization upon stretching shows no age-related difference, which can be attributed to opposite effects of the decrease of collagen content and of the increase of collagen cross-linking in old mice. Aging is a complex process that affects the function of all organs and tissues and most often has an irreversible impact on their mechanical behavior. The most visible effects of aging are observed in skin and have been extensively studied for medical and cosmetic purposes. The three skin layers are affected both structurally and functionally. However, aging primary impacts the mechanical integrity of the dermis. At macroscopic scale, the mechanical behavior of aged dermis shows an increased stiffness and a decreased ability to recoil 1–3. At lower scales, a complex multi-parameters process eventually results in a decrease of collagen and elastin contents due to an imbalance between matrix proteins synthesis and degradation by matrix metalloproteinases, an increase of collagen cross-linking, a deterioration of proteoglycans and a subsequent loss of water 4–9. However, the link between these microstructural modifications and the mechanical changes has so far been inferred rather than experimentally demonstrated due to the technical issues encountered when trying to obtain multiscale data. Collagens are the main component of the dermis and other connective tissues 7,10. Fibril-forming collagens assemble into striated fibrils, the diameter and three-dimensional organization of which are tissue-specific. They form multiprotein networks with other matrix proteins such as the elastin fibers and non-fibrillar matrix (pro-teoglycans, glycoaminoglycans…) that determine the mechanical behavior of dermis and other collagen-rich tissues 11–18. Collagen fibers are usually heterotypic structures. In dermis, they are made of type I, III and V col-lagens. Type V collagen is a minor component that acts as a regulatory fibril-forming collagen 19,20. As such, it plays an important role in the pathogenesis of the classical Ehlers-Danlos (EDS) syndrome. This rare connective tissue disease illustrates the close link between collagen microstructure and tissue mechanics since it is caused by mutations in collagen V genes, while being primary characterized by skin hyperextensibility 19–21. Moreover, EDS patients show a prematurely aged skin, which illustrates the close link between collagen microstructure and skin aging.