Lifestyle and genetic factors can lead to the development of atherosclerosis and, ultimately, cardiovascular adverse events. Rodent models are commonly used to investigate mechanism(s) of atherogenesis. However, the 3Rs principles, aiming to limit animal testing, encourage the scientific community to develop new physiologically relevant in vitro alternatives. Leveraging the 96-chip OrganoPlate(R), a microfluidic platform, we have established a three-dimensional (3D) model of endothelial microvessels-on-a-chip under flow using primary human coronary arterial endothelial cells. As functional readout, we have set up an assay to measure the adhesion of monocytes to the lumen of perfused microvessels. For monitoring molecular changes in microvessels, we have established the staining and quantification of specific protein markers of inflammation and oxidative stress using high content imaging, as well as analyzed transcriptome changes using microarrays. To demonstrate its usefulness in systems toxicology, we leveraged our 3D vasculature-on-a-chip model to assess the impact of the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product, and the 3R4F reference cigarette on the adhesion of monocytic cells to endothelial microvessels. Our results show that THS 2.2 aerosol-conditioned medium had a reduced effect on monocyte-endothelium adhesion compared with 3R4F smoke-conditioned medium. In conclusion, we have established a relevant 3D vasculature-on-a-chip model for investigating leukocyte-endothelial microvessel adhesion. A case study illustrates how the model can be used for product testing in the context of systems toxicology-based risk assessment. The current model and its potential further development options also open perspectives of applications in vascular disease research and drug discovery.