Nutrition in pregnancy and early childhood affects later blood pressure and precursors of atherosclerosis, but its influence on arterial stiffness is unexplored. This study determines whether exposure to improved nutrition during early life influences Augmentation index (AI) and pulse wave velocity (PWV) in mid-adulthood. We included 1221 adults (37-54y) who participated in a cluster-randomized nutritional supplementation trial of a protein-energy beverage (Atole), conducted between 1969-1977 in Guatemala. The comparison group received Fresco, a low-calorie protein-free beverage. In 2015-17, we measured anthropometry (weight, height, and waist-to-height ratio); AI and PWV (using carotid-femoral tonometry); blood pressure; fasting plasma glucose and serum lipids; and sociodemographic characteristics. Based on patterns of exposure, we characterized participants as fully, partially or unexposed to the intervention from conception to their second birthday (the ‘first 1000 days’). We fit pooled and sex-specific models using intention-to-treat, difference-in-difference regression analysis to test whether exposure to the supplement in the first 1000 days was associated with AI and PWV in adulthood adjusting for basal and current sociodemographic variables and current life-style and cardio-metabolic risk factors. Prevalence of obesity in men and women was 39.6% and 19.6%, and prevalence of hypertension was 44.0% and 36.0%, respectively. Women had higher AI (34.4±9.6%) compared to men (23.0± 9.8%), but had similar PWV (7.60±1.13 m/s and 7.60±1.31, respectively). AI did not differ significantly across intervention groups. PWV was lower in individuals with full exposure to the supplement during the first 1000 days (-0.39m/s, 95% CI -0.87, 0.09; p = 0.1) compared to unexposed individuals. This difference was similar after adjusting for cardio-metabolic risk factors (-0.45m/s; 95%C-0.93, 0.01; p = 0.06). Exposure to improved nutrition during the first 1000 days was marginally associated with lower PWV, but not with AI.