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To evaluate the reliability and validity of five commonly used physical activity questionnaires (PAQ) in women aged 45-65 yr with varying physical activity (PA) levels.Data were obtained from the Evaluation of Physical Activity Measures in Middle-aged Women (PAW) Study and included 66 women (aged 52.6 ± 5.4 yr). PAQ evaluated include Modifiable Activity Questionnaire (past week and past month version), Nurses' Health Study PAQ, Active Australia Survey, and Women's Health Initiative PAQ. Intraclass correlation coefficients (ICC) between administrations of the PAQ were used to assess test-retest reliability. Spearman rank-order correlation coefficients were used to examine the associations of PA and physical fitness data with PAQ summary estimates.Accelerometer-determined median (25th, 75th percentiles) times (min·d−1) spent in moderate-lifestyle [760-1951 counts (ct)], moderate-walk (1952-5724 ct), vigorous (≥5725 ct), and combined moderate and vigorous PA (MVPA ≥ 1952 ct) during the 35 d of observation were 66.0 (51.2, 81.3), 23.1 (14.1, 34.6), 0.4 (0.0, 2.3), and 24.3 (15.9, 41.6) min, respectively. The PAQ were shown to be reproducible and relatively stable over time (ICC = 0.32 to 0.91) and were associated with total counts per day (ct·d−1, 0.46 to 0.60, all P < 0.001), and most were associated with many facets of physical fitness, including cardiorespiratory fitness (0.36 to 0.46, P < 0.01), body composition (−0.27 to −0.34, P < 0.05), and muscular fatigue (−0.25 to −0.44, P < 0.05).The PAQ evaluated in this study were shown to be reliable and associated with PA and physical fitness measures. Current findings support the utility of these PAQ for PA assessment in research studies of middle-aged women.