Exposure to airborne particulate matter (PM) has been attributed to millions of deaths annually. However, the PM components responsible for observed health effects remain unclear. Oxidative potential (OP) has gained increasing attention as a key property that may explain PM toxicity. Using online measurement methods that impinge particles for OP quantification within seconds, we reveal that 60 to 99% of reactive oxygen species (ROS) and OP in secondary organic aerosol and combustion-generated PM have a lifetime of minutes to hours and that the ROS activity of ambient PM decays substantially before offline analysis. This implies that current offline measurement methods substantially underestimate the true OP of PM. We demonstrate that short-lived OP components activate different toxicity pathways upon direct deposition onto reconstituted human bronchial epithelia. Therefore, we suggest that future air pollution and health studies should include online OP quantification, allowing more accurate assessments of links between OP and health effects.