To examine whether accelerometer-measured physical activity-based reactivity was present in a nationally representative sample of U.S. children (6-11 yrs), adolescents (12-17 yrs), and adults (≥20 yrs). Data from the 2003-2006 National Health and Nutrition Examination Survey (N = 674, 6-85 yrs) were used. Physical activity (PA) was assessed using the ActiGraph 7164 accelerometer, with PA assessed over 7 days of monitoring. Two PA metrics were assessed, including activity counts per day (CPD) and time spent in moderate-to-vigorous PA. Evidence of reactivity was defined as a statistically significantly change in either of these 2 PA metrics from day 1 of monitoring to days 2 or 3, with day 1 of monitoring being a Monday. Suggestion of reactivity was observed only for the adult population where CPD from days 2 and 3 (297,140.6 ± 7920.3 and 295,812.9 ± 8364.9), respectively, differed significantly from day 1 (309,611.5 ± 9134.9) over the monitoring period (4.0% to 4.5% change). The analysis was conducted 2 additional times with differing start days (Tuesday and Wednesday), and this approach failed to demonstrated a reactive presence. In this national sample of U.S. children, adolescents and adults, we did not observe sufficient evidence of accelerometer reactivity.