Endothelial dysfunction constitutes a hallmark of vascular aging that can have serious implications for disease risk. In addition, aging is associated with mitochondrial dysfunction, which can lead to increased oxidative stress and create associated damages. Time-restricted feeding (TRF) is a dietary intervention known to have many benefits on age-related disease risk. The objective of this study is to elucidate the effects of TRF upon endothelial mitochondrial function of aorta during aging. We hypothesized that TRF protects the aorta from age-related endothelial mitochondrial dysfunction. Adult C57BL/6 mice underwent TRF regimen, including six hours of ad libitum feeding daily. Young and age-matched control mice were fed ad libitum. After 12 months of TRF, wire myography was performed on aorta tissue as a measure of endothelial function. The aorta received pharmacological treatments to induce contraction, and dose-dependent dilation was quantified. For the first time, simultaneous measurement of reactive oxygen species (ROS) and oxygen consumption were completed in aorta tissue using Oroboros O2K Fluorespirometry. Dihydroethidium (DHE) fluorescent staining of ROS in aorta tissue was used to validate ROS measurements from O2K. Serum ketone body levels related to metabolic switching and relevant to mitochondrial biogenesis were determined. TRF improved endothelial-dependent dilation of aorta tissue, representing the intervention’s protection of endothelial function in aging. Aged TRF mice had greater oxygen consumption and less ROS in aorta tissue than age-matched control mice, indicating improved vascular mitochondrial function after TRF intervention. Aged TRF mice had elevated serum levels of ketone bodies involved in mitochondrial biogenesis. This supports the hypothesis that age-related changes in vascular mitochondrial health are alleviated by TRF. The knowledge gained from this study is relevant to understanding the mechanisms of age-related vascular dysfunction. Stephenson Cancer Center, National Institute of Aging R03 AG070479, American Heart Association. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.