Purpose Although physical activity is an established risk factor for chronic disease prevention, the specific mechanisms underlying these relationships are poorly understood. We examined the associations between total activity counts (TAC) and moderate-vigorous physical activity (MVPA) measured by accelerometer, and physical activity energy expenditure (PAEE) measured by doubly-labeled water, with plasma levels of pro-insulin, insulin, c-peptide, IGFBP-3, IGF-1, adiponectin, leptin and leptin-sR. Methods We conducted a cross-sectional analysis of 526 healthy US women in the Women’s Lifestyle Validation Study, 2010–2012. We performed multiple linear regression models adjusting for potential lifestyle and health-related confounders to assess the associations between physical activity, measured in quartiles (Q) and biomarkers. Results Participants in Q4 vs. Q1 of TAC had lower proinsulin (−20%), c-peptide (−7%), insulin (−31%) and leptin (−46%) levels, and higher adiponectin (55%), leptin-sR (25%) and IGF-1 (9.6%) levels (all P-trend≤0.05). Participants in Q4 vs Q1 of MVPA had lower proinsulin (−26%), c-peptide (−7%), insulin (−32%) and leptin (−40%) levels, and higher adiponectin (31%) and leptin-sR (22%) levels (all P-trend≤0.05). Further adjustment for body mass index attenuated these associations, but the associations with adipokines remained significant. Those in Q4 vs. Q1 of PAEE had lower leptin (−21%) and higher leptin-sR (10%) levels (all P-trend ≤0.05), after additional adjustment for body mass index. In the sensitivity analysis, the associations were similar but attenuated when physical activity was measured using the subjective physical activity questionnaire. Conclusion Our data suggests greater physical activity is modestly associated with favorable levels of cardiometabolic and endocrine biomarkers, where the strongest associations were found with accelerometer-measured physical activity. These associations may be only partially mediated through BMI, further supporting the role of physical activity in the reduction of cardiometabolic and endocrine disease risk, independent of adiposity.