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Objective Circadian disruption, as experienced by night shift workers, increases the risk of cardiometabolic diseases. The aim of this study is to investigate metabolite alterations and their relation to cardiometabolic outcomes among night shift workers.Material and Methods Data from exposure measurements, questionnaires including diet, clinical examination (anthropometry including BMI, blood pressure, waist and hip circumference), and biological samples after shift work were collected within the EPHOR-NIGHT project (N=860, day shift n=363, night shift =497) from Sweden, Spain and Denmark. Plasma metabolites (cholesterol, triglycerides, fatty acids, apolipoproteins, HbA1c and fasting blood glucose (the latter two only assessed in the Swedish sub-cohort n=156-169) were quantified by 1 H NMR Spectroscopy and routine clinical assays. Using adjusted linear regression models, including diet, we examined differences in plasma metabolite levels and their association with cardiometabolic risk factors among night shift workers compared with day shift workers.Results No differences were found for total cholesterol, triglycerides, or apolipoproteins in the whole cohort, nor fasting glucose or HbA1c in the sub-cohort. Night-shift workers had lower fractions of PUFAs, both omega-3 (-0.22, 95% CI -0.38 to -0.07) and omega-6 (-0.56, -0.99 to -0.12), and total PUFAs (-0.78, -1.22 to -0.35). Conversely, they had higher fractions of mono-unsaturated (0.48, 0.11 to 0.84) and saturated fatty acids (0.31, 0.06 to 0.55). Lower fractions of PUFAs and higher fractions of mono- and saturated fatty acids metabolites fractions were in turn significantly associated with higher BMI and higher blood pressure. Further mediation and dose-response analyses will be presented at the conference.Conclusions We found differences in potential metabolic pathways linking night shift work with cardiometabolic risk. Contributions from other e.g. behavioral and environmental factors cannot be ruled out and warrant further longitudinal investigation.Funding Research funding from the EU Horizon project EPHOR, Swedish Research Council FORTE and AFA insurance.