The mechanical properties of human aortas are linked to the layered tissue and its microstructure at different length scales. Each layer has specific mechanical and structural properties. While the ground substance and the elastin play an important role in tissue stiffness at small strain, collagen fibers carry most of the load at larger strains, which corresponds to the physiological conditions of the aorta at maximum pulsatile blood pressure. In fact, collagen fibers are crimped in the unloaded state. Collagen fibers show different orientation distributions when they are observed in a plane that is tangent to the aortic wall (in-plane section) or along a direction orthogonal to it (out-of-plane section). This was systematically investigated using large images (2500 × 2500 µm) with high resolution obtained by second harmonic generation (SHG) in order to homogenize tissue heterogeneity after a convergence analysis, which is a main goal of the study. In addition, collagen fibers show lateral interactions due to entanglements and the presence of transverse elastin fibers, observed on varying length scales using atomic force microscopy and a three-dimensional rendering obtained by stacking a sequence of SHG and two-photon fluorescence images; this is another important contribution. Human descending thoracic aortas from 13 heartbeat donors aged 28 to 66 years were examined. Uniaxial tensile tests were carried out on the longitudinal and circumferential strips of the aortic wall and the three separated layers (intima, media and adventitia). A structurally-motivated material model with (i) a term to describe the combined response of ground substance and elastin and (ii) terms to consider four families of collagen fibers with different directions was applied. The exclusion of compressed fibers was implemented in the fitting process of the experimental data, which was optimized by a genetic algorithm. The results show that a single fiber family with directional and dispersion parameters measured from SHG images can describe the mechanical response of all 39 layers (3 layers for each of the 13 aortas) with very good accuracy when a second (auxiliary) family of aligned fibers is introduced in the orthogonal direction to account for lateral fiber interaction. Indeed, all observed distributions of collagen directions can be accurately fitted by a single bivariate von Mises distribution. Statistical analysis of in-plane and out-of-plane dispersion of fiber orientations reveals structural differences between the three layers and a change of collagen dispersion parameters with age. STATEMENT OF SIGNIFICANCE: The stiffness of healthy young aortas is adjusted so that a diameter expansion of about 10 % is possible during the heartbeat. This creates the Windkessel effect, which smooths out the pulsating nature of blood flow and benefits organ perfusion. The specific elastic properties of the aorta that are required to achieve this effect are related to the microstructure of the aortic tissue at different length scales. An increase in the aortic stiffness, in addition to reducing cyclic expansion and worsening perfusion, is a risk factor for clinical hypertension. The present study relates the microstructure of healthy human aortas to the mechanical response and examines the changes in microstructural parameters with age, which is a key factor in increasing stiffness.