Objective: The aim of this study was to evaluate the relationship between objectively measured physical activity (ACT), particularly intensity of physical activity (iACT), and body fat percentage (BF%) in 278 middle-aged women. Secondary purposes were to ascertain the association between physical activity duration (dACT) and BF% and the extent to which potentially confounding factors, including total ACT, energy intake, body weight, and season of assessment, influenced the relationship between iACT and BF%. Research Methods and Procedures: A cross-sectional design was used. Subjects were apparently healthy, 35 to 45 years old, premenopausal, nonsmokers, with BMIs < 30 kg/m(2). Approximately 90 percent were white, 81% were married, and 37% were college graduates. ACT was assessed using Computer Science and Application accelerometers worn for 7 consecutive days. The accelerometers recorded movement continuously, and activity counts were collapsed into 10-minute epochs. Intensity was indexed using seven activity count cut-off points, and duration was based on the number of 10-minute epochs at each intensity level. BF% was assessed using multiple measurements in the Bod Pod. Energy intake was measured using 7-day weighed diet records during the same week subjects wore the accelerometers. Results: BF% was strongly and inversely associated with iACT and dACT. Controlling for energy intake and body weight strengthened the relationships among iACT, dACT, and BF%. Control of total ACT weakened the association. Discussion: Engaging in higher intensity and/or longer duration ACT is associated with lower BF% compared with lower intensity and/or shorter durations of activity.