Background: The 400 m walk test has been used in older adults; however, the applicability in middle-aged populations is unknown. Methods: Data were obtained from the Evaluation of Physical Activity Measures in Middle-Aged Women (PAW) Study and included 66 women (52.6 ± 5.4 years). Participants were instructed to walk at a brisk, maintainable pace; time taken to complete the 400 m was recorded in seconds. Intraclass correlation coefficients (ICC) were used to assess test-retest reliability. Spearman rank order correlation coefficients were used to examine the concurrent validity of the walk test with cardiorespiratory fitness and associations with physical activity, body composition, flexibility, static balance, and muscular fitness, adjusted for age and body mass index. Results: Participants completed the walk at visits 4 and 5 in 248.0 and 245.0 seconds, respectively. The walk test had excellent reproducibility [ICC = 0.95 (95% CI: 0.92, 0.97)] and was significantly associated with estimated (ρ = -0.43; P < 0.0001) and measured (ρ = -0.56; P < 0.001) VO2max. The walk test was also significantly related to physical activity, body composition, flexibility, and balance. Conclusions: These findings support the utility of the 400 m walk test to estimate cardiorespiratory fitness and reflect free-living physical activity in healthy, middle-aged women. © 2010 Human Kinetics, Inc.