Project Summary Age is the primary risk factor for cardiovascular disease (CVD) and age-related vascular dysfunction is considered the key process linking the two. Middle age is particularly vulnerable period when risk factors exceed diagnostic thresholds and clinical expression of CVD first becomes evident. Ultra-processed foods (UPF) comprise almost 60% of total energy in the standard American diet (SAD). The results of observational studies suggest that UPF consumption increases CVD risk, independent of overall diet quality (i.e., saturated fat, sodium, sugar, and dietary fiber intake). The “industrialized microbiota” may link diet, particularly UPF, to increased inflammation and CVD in middle-aged adults. High intake of UPF increases the likelihood of an excess heart age >10years and doubles the risk of subclinical coronary atherosclerosis in middle-aged adults. However, the impact of reducing UPF consumption on vascular function in middle-aged adults is unknown. The overall objective of this R21 proposal is to establish proof-of-concept for an improvement in vascular function following reductions in UPF consumption in middle-aged adults, in order to conduct a larger, more comprehensive and mechanistic trial in the future. To this end, following a 2-week eucaloric lead-in diet (SAD with 60% UPF), 34 healthy middle-aged adults (45-64 yrs) will complete a 4-week randomized crossover trial of both a SAD and an un/minimally processed foods (0% UPF). A 2-week washout period (same SAD lead-in diet) will separate the treatments. Participants will be fed a eucaloric diet (50% carbohydrate, 35% fat, 15% protein) matched for dietary fiber, added sugars, saturated fat, mono- and poly-unsaturated fat, sodium, pre- and probiotics, micronutrients, and overall diet quality, for the duration of the study to avoid potential confounds of weight change and other dietary factors which may influence study outcomes. Commercial additive content of study diets will be quantified, and study diet composition will be verified by chemical analysis. Measurements of flow- mediated dilation, arterial stiffness, gut microbiota composition/function, fecal short chain fatty acids (SCFA), intestinal inflammation, intestinal permeability, serum endotoxin, and inflammatory cytokines will be made before and following each 4-week treatment period. This innovative study will be conducted by an established investigative team with extensive experience and a strong record of success performing well-controlled dietary intervention studies targeting aging and CVD risk.