BACKGROUND: Weight loss is associated with improvements in cardiometabolic risk factors, including serum glucose, insulin, C-reactive protein, and blood lipids. Few studies have evaluated the long-term (>18 months) effect of weight loss on these risk factors or sought to identify factors associated with sustained improvements in these measures. METHODS AND RESULTS: In 417 overweight/obese women (mean [SD] age, 44  years) participating in a weight loss trial, we sought to identify predictors of weight loss-associated cardiometabolic risk factors after 12 and 24 months of intervention. Total cholesterol (TC), low-density lipoprotein (LDL)-cholesterol (LDL-C), high-density lipoprotein (HDL)-cholesterol, non-HDL-cholesterol, triglycerides (TG), insulin, glucose, C-reactive protein (CRP), and cardiopulmonary fitness were measured at baseline and at 12 and 24 months. After 24 months, significant reductions in body weight, waist circumference, CRP, TC, HDL-cholesterol, and non-HDL-cholesterol were observed (P<0.01). After 24 months, mean TC and non-HDL-cholesterol were reduced regardless of the amount of weight lost, whereas reductions in LDL-cholesterol, CRP, insulin, and TG were observed only in those who lost >/=10% body weight. Step-test performance improved only in those who lost >/=10% body weight after 24 months. Change in weight demonstrated a positive predictive value for change in cholesterol, insulin, glucose, and triglycerides. Baseline level of the biomarker showed the greatest predictive value for follow-up measures for insulin, cholesterol, glucose, and triglycerides. CONCLUSIONS: Our data extend the results from short-term weight loss trials and suggest that the magnitude of weight loss and baseline values for risk factors are associated with improvements in cardiometabolic risk factors even after 24 months.