We recently showed that Western diet–induced obesity and insulin resistance promotes endothelial cortical stiffness in young female mice. Herein, we tested the hypothesis that regular aerobic exercise would attenuate the development of endothelial and whole artery stiffness in female Western diet–fed mice. Four-week-old C57BL/6 mice were randomized into sedentary (ie, caged confined, n=6) or regular exercise (ie, access to running wheels, n=7) conditions for 16 weeks. Exercise training improved glucose tolerance in the absence of changes in body weight and body composition. Compared with sedentary mice, exercise-trained mice exhibited reduced endothelial cortical stiffness in aortic explants (sedentary 11.9±1.7 kPa versus exercise 5.5±1.0 kPa; P<0.05), as assessed by atomic force microscopy. This effect of exercise was not accompanied by changes in aortic pulse wave velocity (P>0.05), an in vivo measure of aortic stiffness. In comparison, exercise reduced femoral artery stiffness in isolated pressurized arteries and led to an increase in femoral internal artery diameter and wall cross-sectional area (P<0.05), indicative of outward hypertrophic remodeling. These effects of exercise were associated with an increase in femoral artery elastin content and increased number of fenestrae in the internal elastic lamina (P<0.05). Collectively, these data demonstrate for the first time that the aortic endothelium is highly plastic and, thus, amenable to reductions in stiffness with regular aerobic exercise in the absence of changes in in vivo whole aortic stiffness. Comparatively, the same level of exercise caused destiffening effects in peripheral muscular arteries, such as the femoral artery, that perfuse the working limbs.