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Chronically increased levels of high molecular weight advanced glycation end products (HMW-AGEs) are known to induce cardiovascular dysfunction. Whether an acute increase in HMW-AGE levels affects vascular function remains unknown. In this study, we examined whether acute exposure to HMW-AGEs disturbs aortic vasomotor function. Aortae were obtained from healthy male rats and were acutely pre-treated with HMW-AGEs in organ baths. Aortic relaxation responses to cumulative doses of acetylcholine (ACh), in the presence or absence of superoxide dismutase (SOD), were measured after precontraction with phenylephrine (PE). Furthermore, levels of 3-nitrotyrosine were evaluated on aortic paraffine sections. In our study, we show that acute exposure to HMW-AGEs significantly decreases the aortic relaxation response to ACh. SOD pre-treatment prevents acute HMW-AGEs-induced impairment by limiting superoxide formation. In conclusion, our data demonstrate that acute exposure to HMW-AGEs causes adverse vascular remodelling, characterised by disturbed vasomotor function due to increased oxidative stress. These results create opportunities for future research regarding the acute role of HMW-AGEs in cardiovascular dysfunction. This research was funded by a Bijzonder onderzoeksfonds (BOF) grant from Hasselt University, grant number: 16NI05BOF. We thank Jeanine Santermans for embedding the tissues in paraffin. The authors also thank Petra Bex for her skilful technical assistance. The graphical abstract was created using images from Biorender and ServierMedical Art Commons Attribution 3.0 Unported License (http: //smart.servier.com, accessed on 2 September 2022). Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License.