Background: Accelerometers have emerged as a useful tool for measuring free-living physical activity in epidemiological studies. Validity of activity estimates depends on the assumption that measurements are equivalent for males and females while performing activities of the same intensity. The primary purpose of this study was to compare accelerometer count values in males and females undergoing a standardized 6-minute walk test. Methods: The study population was older adults (78.6 ± 4.1 years) from the AGES-Reykjavik Study (N = 319). Participants performed a 6-minute walk test at a self-selected fast pace while wearing an ActiGraph GT3X at the hip. Vertical axis counts•s -1 was the primary outcome. Covariates included walking speed, height, weight, BMI, waist circumference, femur length, and step length. Results: On average, males walked 7.2% faster than females (1.31 vs. 1.22 m•s -1, P < .001) and had 32.3% greater vertical axis counts•s -1 (54.6 vs. 39.4 counts•s -1, P < .001). Accounting for walking speed reduced the sex difference to 19.2% and accounting for step length further reduced the difference to 13.4% (P < .001). Conclusion: Vertical axis counts•s -1 were disproportionally greater in males even after adjustment for walking speed. This difference could confound free-living activity estimates.