Oxidative stress is one hypothesized mechanism linking anthropometric, behavioral, and medical risk factors with cardiovascular disease (CVD). We evaluated cross-sectional associations between CVD risk factors and biomarkers of oxidative stress, and investigated these biomarkers as predictors of incident diabetes and hypertension among premenopausal women. F2-isoprostane (F2-IsoP) and metabolite (15-F2t-IsoP-M), reliable biomarkers of oxidative stress, were measured in urine samples collected at enrollment from 897 premenopausal women (ages 35–54) enrolled in the Sister Study cohort without a CVD history. Blood pressure, waist circumference, and body mass index (BMI) were measured at enrollment by trained study personnel. Diabetes and cigarette smoking were self-reported via enrollment questionnaires. Over a maximum follow-up of 11.5 years, participants self-reported incident diabetes and hypertension diagnoses on mailed questionnaires. In cross-sectional analyses, both F2-IsoP and 15-F2t-IsoP-M were positively associated with BMI, waist circumference, diastolic blood pressure, and current smoking. F2-IsoP was elevated among those with diabetes, and 15-F2t-IsoP-M increased with higher systolic blood pressure. Prospective analyses suggested an increased hypertension risk among those with elevated 15-F2t-IsoP-M (highest vs. lowest quartile: hazard ratio=2.34; 95% CI: 1.20–4.56). Our results suggest that urinary F2-IsoP and 15-F2t-IsoP-M are positively associated with adiposity measures, blood pressure, and cigarette smoking. Further investigation is warranted to evaluate 15-F2t-IsoP-M as a predictor of hypertension.