Background/Aim:Short-term exposures to air pollution and temperature have been reported to be associated with inflammation and oxidative stress. However, mechanistic understanding of the affected metabolic pathways is still lacking and the existing literature on the short-term exposure of air-pollution on the metabolome is limited. We aimed to determine the changes in the blood metabolome and the associated pathways related to short-term exposure to outdoor air pollution and temperature. Methods:We performed mass-spectrometry based untargeted metabolomic profiling of plasma samples from a large and well-characterized cohort of men (the Normative Aging Study) to identify metabolic pathways associated with short-term exposure to PM2.5, NO2, O3, and temperature (one, seven-, and thirty-day average of central site monitors). We used multivariable linear mixed-effect regression and independent component analysis (ICA) while simultaneously adjusting for all exposures and correcting for multiple testing. Results:Overall, 456 white men provided 648 blood samples, in which 1,158 metabolites were quantified, between 2000 and 2016. Average age and body mass index were 75.0 years and 27.7 kg/m2, respectively. Only 3% were current smokers. In the adjusted models, PM2.5, NO2, and temperature showed multiple statistically significant associations with several metabolites (40 metabolites for PM2.5, 100 metabolites for NO2, 1 metabolite for O3, and 14 metabolites for temperature). We identified eight metabolic pathways (glycerophospholipid, sphingolipid, glutathione, beta-alanine, pyrimidine, butanoate, propanoate, and methionine, cysteine, SAM and taurine metabolisms) perturbed with short-term exposure to air pollution and temperature. These pathways were involved in inflammation and oxidative stress, immunity, and nucleic acid damage and repair. Conclusions:We identified several significant metabolites and metabolic pathways associated with short-term exposure to air pollution and temperature; using an untargeted approach. This is the first study to report an untargeted metabolomic signature of temperature exposure, the first to use ICA, and the largest study to derive a metabolomic signature of air pollution.