Objective: Despite the well-supported biological link between physical activity (PA) and atherosclerosis, most previous studies have reported a null association between PA and coronary artery calcification (CAC). The aim of this study was to examine the relationship between PA and CAC progression in 148 Healthy Women Study (HWS) participants over 28 years of observation. Methods: The HWS was designed to examine cardiovascular risk factor changes from premenopause to postmenopause. Based on CAC scores collected on two follow-up visits (electron beam tomography [EBT] 1 and EBT4) scheduled 12 years apart, participants were classified into one of three groups: (1) no-detectable CAC group (n = 37; 0 CAC on both visits); (2) incident CAC group (n = 46; 0 CAC on the first visit and >0 CAC on the last visit); or (3) prevalent CAC group (n = 65; >0 CAC on both visits). PA data were collected regularly throughout the study using self-report questionnaires and accelerometers on EBT4. Results: The percentage of HWS participants with no detectable CAC decreased from 56.1% on EBT1 to 25.0% on EBT4. Times spent per day in accumulated moderate-to vigorous-intensity PA (MVPA) and bouts of MVPA were each significantly higher in the no-detectable CAC group when compared with the prevalent CAC group (both P ≤ 0.01). After covariate adjustment, these differences remained statistically significant (both P < 0.05). Although self-reported summary estimates collected throughout the study were significantly associated with accelerometer data on EBT4, there were no significant differences in self-reported PA levels by CAC group after covariate adjustment. Conclusions: Study findings suggest that low levels of accelerometer-derived MVPA may be indicative of subclinical disease in older women. © 2012 by The North American Menopause Society.