Introduction: Occupational physical activity (OPA) has been linked to adverse cardiovascular outcomes and mortality among workers, but underlying mechanisms are not clear. Allostatic load (AL) presents a measure of the accumulative burden of chronic stress and may play a role in the association of OPA with cardiovascular outcomes. We hypothesized that OPA was positively associated with AL in workers. Methods: Analyses were conducted in 6487 United States (U.S.) workers (i.e., working at a job or business) aged 20-60 years who were not pregnant, had data on moderate-to-vigorous OPA and biomarkers for AL calculation using a national representative sample, the National Health and Nutrition Examination Survey (2007-2018). OPA was dichotomized primarily by time spent on moderate-to-vigorous OPA (i.e., high: ≥ 150 vs. low: < 150; min/week) and secondarily by energy spent on it (i.e., high: ≥ median vs. low: < median; kcal/week). AL score was calculated using a common method from 9 biomarkers with a range of 0-9. Associations of OPA with AL were assessed using multivariable linear regression models with sampling weights applied to represent U.S. workers. Results: AL score was significantly higher among workers who spent higher time on moderate-to-vigorous OPA (β = 0.11, P = 0.02) or had higher energy on it (β = 0.10, P = 0.05) than their counterparts. When stratified by gender and age, the positive associations of OPA with AL remained significant only among females aged 20-40 years (β = 0.16, P = 0.02 for higher time on moderate-to-vigorous OPA; β = 0.35, P < 0.001 for higher energy on moderate-to-vigorous OPA), but such associations were not observed in other groups (females aged 41-60 years, males aged 20-40 years, and males aged 41-60 years). Conclusions: We found that OPA was positively associated with AL among U.S. workers, particularly in young female workers. Our findings are novel and worthy of further studies to confirm the role of OPA in explaining cardiometabolic disorders.