...Here, we developed a novel, translational experimental model that allows for direct interrogation of the influence of the circulating milieu on age-related arterial dysfunction (aortic stiffening; endothelial dysfunction). To do so, we exposed young and old mouse arteries to serum from young and old mice and young and mid-life/older (ML/O) adult humans. We found that old mouse and ML/O adult human, but not young, serum stiffened young mouse aortic rings, assessed via elastic modulus (mouse and human serum: p=0.003 vs. young serum control), and impaired carotid artery endothelial function, assessed by endothelium-dependent dilation (EDD) (mouse serum: p<0.001; human serum: p=0.006 vs. young serum control). Furthermore, young mouse and human, but not old, serum reduced aortic elastic modulus (mouse serum: p=0.009; human serum: p<0.001 vs. old/MLO serum control) and improved EDD (mouse and human serum: p=0.015 vs. old/MLO serum control) in old arteries. In human serum-exposed arteries, in vivo arterial function assessed in the human donors correlated with circulating milieu-modulated arterial function in young mouse arteries (aortic stiffness: r=0.634, p=0.005; endothelial function: r=0.609, p=0.004) and old mouse arteries (aortic stiffness: r=0.664, p=0.001; endothelial function: r=0.637, p=0.003). This study establishes novel experimental approaches for directly assessing the effects of the circulating milieu on arterial function and implicates changes in the circulating milieu as a mechanism of in vivo arterial aging.