The purpose of this study was to examine the importance of midlife physical activity on physical functioning in later life. Data are from 1771 Study of Women's Health Across the Nation (SWAN) participants, aged 42–52 (46.4 ± 2.7) years at baseline (1996–97). Latent class growth analysis was used to identify physical activity trajectory groups using reported sports and exercise index data collected at seven time-points from baseline to Visit 13 (2011–13); objective measures of physical functioning performance were collected at Visit 13. The sports and exercise index (henceforth: physical activity) is a measure of moderate to vigorous intensity physical activity during discretionary periods of the day. Multivariable linear regression analyses were used to model each continuous physical performance measure as a function of the physical activity trajectory class. Across midlife, five physical activity trajectory classes emerged, including: lowest (26.2% of participants), increasing (13.4%), decreasing (22.4%), middle (23.9%), and highest (14.1%) physical activity. After full adjustment, women included in the middle and highest physical activity groups demonstrated ≥ 5% better physical functioning performance than those who maintained low physical activity levels (all comparisons; p < 0.05). Statistically significant differences were also noted when physical activity trajectory groups were compared to the increasing physical activity group. Results from the current study support health promotion efforts targeting increased (or maintenance of) habitual physical activity in women during midlife to reduce future risk of functional limitations and disability. These findings have important public health and clinical relevance as future generations continue to transition into older adulthood.