BackgroundAging and vitamin D deficiency have been associated with reduced nitric oxide (NO) synthesis and impaired endothelial function (EF) but the evidence in humans remains weak. ObjectivesTwo independent cross-sectional studies were designed to evaluate the association between age, sex, and plasma vitamin D concentrations with physiological and biochemical biomarkers of NO synthesis and EF in young and older healthy participants (Study 1) and in overweight and obese postmenopausal females (Study 2). MethodsIn Study 1, 40 young (20–49 y) and older (50–75 y) males and females (10 participants per age and sex group) were included. Resting blood pressure and ear-to-finger peripheral pulse wave velocity (PWV) were measured. A stable-isotopic method was used to determine whole-body NO production. Plasma 25-hydroxyvitamin D (25(OH)D), nitrate, nitrite, and asymmetric dimethylarginine (ADMA) concentrations were determined. In Study 2, 80 older overweight and obese females (age 61.2 ± 6.2 y, body mass index 29.5 ± 4.4 kg/m2) were recruited. Postocclusion reactive hyperemia (PORH) and peripheral PWV were measured. Plasma concentrations of 25(OH)D, nitrate, cyclic guanosine monophosphate, 3-nitrotyrosine (3-NT), endothelin-1, vascular endothelial growth factor, and ADMA were determined. ResultsIn Study 1, whole-body NO production was significantly greater in young compared with older participants (0.61 ± 0.30 μmol·h−1·kg−1 compared with 0.39 ± 0.10 μmol·h−1·kg−1, P = 0.01) but there was no evidence of a sex difference (P = 0.81). Plasma 25(OH)D concentration was not associated with PWV (r = 0.18, P = 0.28) or whole-body NO production (r = −0.20, P = 0.22). Plasma ADMA concentration was associated positively with age (r = 0.35, P = 0.03) and negatively with whole-body NO production (r = −0.33, P = 0.04). In Study 2, age was associated with lower PORH (r = −0.28, P = 0.02) and greater ADMA concentrations (r = 0.22, P = 0.04). Plasma 25(OH)D concentration was inversely associated with 3-NT concentrations (r = −0.31, P = 0.004). ConclusionsOlder age was associated with lower whole-body NO production. Plasma vitamin D concentrations were not associated with NO production or markers of EF but showed a weak, significant correlation with oxidative stress in postmenopausal overweight females.