Cardiorespiratory fitness is an independent predictor of cardiovascular events in women; however, maximal oxygen consumption (VO(2max)) protocols are not always feasible. The Evaluation of Physical Activity Measures in Middle-Aged Women Study was a 6-week study designed to evaluate six physical activity (PA) measures in women, aged 45-65 years. We investigated the Ebbeling submaximal treadmill (EbbTM) protocol's ability to reflect an active lifestyle in 66 women [median (IQR) age, 51.0 (48.0, 56.5) years]. Participants were screened using ACSM and AHA guidelines and classified as moderate or low cardiovascular risk. VO(2max) was estimated using the EbbTM in all participants (TEST1). Moderate risk women repeated the EbbTM (TEST2) while low risk completed a maximal treadmill test. Intraclass correlation coefficients (ICC) and minimal detectable change (MDC(90)) score were calculated to examine reliability. Spearman rank order or Pearson product-moment correlation coefficients were used to determine the association between estimated VO(2max) and physical fitness and activity measures. Estimated VO(2max) from the EbbTM protocol had excellent reproducibility [ICC = 0.95 (95% CI: 0.89, 0.98) and MDC(90) was 1.62 mL kg(-1) min(-1). The EbbTM protocol was associated with measured VO(2max) (ρ = 0.48; p = 0.01) and was positively correlated with moderate- (ρ = 0.32; p = 0.02), vigorous- (ρ = 0.47; p = 0.0005) and moderate-to-vigorous-intensity PA (ρ = 0.36; p = 0.009), pedometer steps (ρ = 0.41; p = 0.002) and inversely correlated with waist circumference (ρ = -0.58; p < 0.0001), body fat (ρ = -0.55; p < 0.0001), and right leg endurance (ρ = -0.41; p = 0.003). The EbbTM protocol was shown to be reliable and associated with objective measures of fitness and PA in healthy, middle-aged women.