Background: Extracellular vesicles (EVs) are submicron membrane-bound vesicles released from almost all cells, which affect many pathophysiological processes involved in cardiovascular diseases (CVDs) and may therefore be considered as a novel marker for CVDs. N-3 polyunsaturated fatty acids (n-3 PUFA) have been suggested to play a role in protecting cardiovascular health. However, there is little information about the effect of n-3 PUFA on circulating EV numbers, composition and functional activity in the context of CVDs. Objective: This project investigated the relationships between circulating EVs, cardiovascular risk markers and CVD risk, as well as the chronic effects of fish oil derived n-3 PUFA on the numbers, fatty acid composition and procogulatory activity of circulating EVs. Design: Subjects (n=40) aged 40-70yrs with moderate risk of CVDs were recruited into a double-blind, randomised crossover trial of fish oil (1.8g/d n-3 PUFA) or control oil (high-oleic safflower oil) for 12 weeks with a 12-week washout. EVs were analysed by Nanoparticle Tracking Analysis (NTA) and fluorescence flow cytometry (FCM). Total lipid fatty acid composition and procoagulatory activity of circulating EVs were analysed by gas chromatography and a thrombin generation assay, respectively. Results: Circulating EV numbers were positively associated with body mass index (BMI), blood pressure (BP), plasma triacylglycerol (TAG) concentration and overall CVD risk. Furthermore, plasma TAG concentration and diastolic blood pressure (DBP) independently predicted total EV numbers based on a multivariate regression model. Fish oil supplementation significantly decreased numbers of circulating EVs, modified their total lipid fatty acid composition and decreased their thrombogenic potential. Conclusion: Circulating EVs are associated with CVD risk and are particularly strongly related with plasma TAG concentration and DBP. They could therefore potentially serve as a novel biomarker for CVD prediction. Dietary n-3 PUFA modifies the numbers, fatty acid composition and function of circulating EVs in a manner which suggests beneficial effects of n-3 PUFA on CVDs.