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Exercise improves cardiovascular and metabolic health in pregnancy and may represent a non-pharmacological approach to improving pregnancy outcomes. Extracellular vesicles (EVs) are emerging biomarkers of endothelial dysfunction and offer the potential for evaluating vascular health non-invasively during pregnancy. The purpose of this study was to investigate changes in circulating EV levels after an acute bout of moderate-intensity aerobic exercise in healthy pregnant and non-pregnant women. We studied plasma samples from pregnant (N = 13, 13–28 weeks) and non-pregnant (N = 17) women. A pre-exercise blood sample was obtained followed by a 30 min bout of moderate-intensity treadmill-based exercise. Immediately following the exercise, a post-exercise blood draw was collected. Large EVs were isolated from plasma by differential centrifugation and characterized by Western blot and electron microscopy. We quantified circulating EVs by nanoscale flow cytometry. Endothelial EVs were identified as VE-Cadherin+, platelet EVs as CD41+, and leukocyte EVs as CD45+ events. Acute exercise was associated with a significant reduction in levels of circulating endothelial EVs in the non-pregnant group (p = 0.0232) but not in the pregnant group (p = 0.2734). A greater proportion of non-pregnant women (13/17, 76.47%) exhibited a reduction in endothelial EVs compared with their pregnant counterparts (4/13, 30.76%, p < 0.05). We also observed a positive association between measures of fitness (average speed) and baseline levels of platelet (r = 0.5816, p = 0.0159) and total EVs (r = 0.5325, p = 0.0296) in the non-pregnant group but not in pregnant individuals. Collectively, our study highlights that after a matched acute exercise, changes to circulating EV levels differ depending on pregnancy status. [ABSTRACT FROM AUTHOR]