Frequent and highly aerobic behaviors likely contribute to naturally occurring stress, accelerate senescence and limit lifespan. To understand how the physiological and cellular mechanisms that determine the onset and duration of senescence are shaped by behavioral development and behavioral duration, we exploited the tractability of the honey bee (Apis mellifera) model system. First, we determined whether a cause-effect relationship exists between honey bee flight and oxidative stress by comparing oxidative damage accrued from intense flight bouts to damage accrued from d-galactose ingestion, which induces oxidative stress and limits lifespan in other insects. Second, we experimentally manipulated the duration of honey bee flight across a range of ages to determine the effects on reactive oxygen species (ROS) accumulation and associated enzymatic antioxidant protective mechanisms. In bees fed d-galactose, lipid peroxidation (assessed by measuring malondialdehyde levels) was higher than in bees fed sucrose and age-matched bees with a high and low number of flight experiences collected from a colony. Bees with high amounts of flight experience exhibited elevated 8-hydroxy-2'-deoxyguanosine, a marker of oxidative DNA damage, relative to bees with less flight experience. Bees with high amounts of flight experience also showed increased levels of pro-oxidants (superoxide and hydrogen peroxide) and decreased or unchanged levels of antioxidants (superoxide dismutase and catalase). These data implicate an imbalance of pro- to anti-oxidants in flight-associated oxidative stress, and reveal how behavior can damage a cell and consequently limit lifespan.