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Chronic physical activity may alter estrogen metabolism, a proposed biomarker of breast cancer risk, by causing a shift toward higher 2-OHE1 and lower 16α-OHE1 levels.To investigate the association between an objective indicator of chronic exercise, aerobic fitness, and 2-OHE1 and 16α-OHE1 in premenopausal women.Women with high aerobic fitness (N = 17; O2max ≥ 48 mL·kg·min−1) were compared with women with average aerobic fitness (N = 13; O2max ≤ 40 mL·kg·min−1) in terms of 2-OHE1 and 16α-OHE1 profiles. Participants were healthy, regularly menstruating, Caucasian women, aged 20–42 yr, with a normal body mass index (BMI) of 18–24, not using pharmacologic contraceptives. We measured height, weight, sum of four skinfolds, and maximal aerobic fitness (O2max), using an incremental cycle ergometer test. Urine samples were collected during the follicular and luteal phase of the menstrual cycle.There were no statistically significant differences between average and highly fit women for 2-OHE1, 16α-OHE1, or the 2:16α-OHE1 ratio in either the follicular or luteal phase. However, the high-fitness group showed a trend toward a higher luteal 2:16α-OHE1 (P = 0.20). In ancillary analyses, a higher sum of skinfolds was associated with significantly higher luteal 16-OHE1 levels (r = 0.39, P = 0.03) and lower luteal phase 2:16 OHE ratio (r = −0.41, P = 0.02). Higher BMI was associated with lower follicular phase 2-OHE1 (r = −0.37, P = 0.04) and lower follicular 2:16 OHE1 ratio (r = −40, P = 0.03).This exploratory study is the first to investigate the association between aerobic fitness and estrogen metabolites in premenopausal women using metabolic parameters. We observed no statistically significant association between aerobic fitness and 2-OHE1 and 16α-OHE1, but found that body composition was associated with 2-OHE1 and 16α-OHE1 levels.