Few studies have reported longitudinal relationships between physical activity (PA) and cardiometabolic risk factors over time using repeated assessments in overweight or obese adults. We conducted a longitudinal study in 127 participants (81% with body mass index > 30 kg/m(2)) who completed a 12-month behavioral intervention for weight loss between 2003 and 2005 in Pittsburgh, PA, USA. Using absolute change scores from baseline to each time point (i.e., 6 and 12 months) for all studied variables (Delta = time point - baseline), we performed mixed effects modeling to examine relationships between PA and cardiometabolic risk factors, after adjusting for body weight, energy intake and other covariates (i.e., age, gender, and ethnicity). PA was assessed as energy expenditure (kcal/week) using the Paffenbarger activity questionnaire. Over the 12-month period, energy expenditure increased (Delta1,370 kcal/week at 6 months vs. Delta886 kcal/week at 12 months); body weight decreased (Delta8.9 kg at 6 months vs. Delta8.4 kg at 12 months). The average increase in energy expenditure over 12 months was significantly and independently related to reductions in total cholesterol (F = 6.25, p = 0.013), low-density lipoprotein cholesterol (LDL-C) (F = 5.08, p = 0.025) and fasting blood glucose (F = 5.10, p = 0.025), but not to other risk factors (i.e., fasting insulin, high-density lipoprotein cholesterol, triglycerides, and waist circumference). In conclusion, among overweight and obese adults undergoing a weight loss intervention, increased energy expenditure over 12 months may improve total cholesterol and LDL-C, important coronary risk factors, and fasting blood glucose, a metabolic risk factor.