Cardiovascular diseases (CVDs) in the course of atherosclerosis are one of the most critical public health problems in the world. Endothelial cells synthesize numerous biologically active substances involved in regulating the functions of the cardiovascular system. Endothelial dysfunction is an essential element in the pathogenesis of atherosclerosis. Thus, the assessment of endothelial function in people without overt CVD allows for a more accurate estimate of the risk of developing CVD and cardiovascular events. The assessment of endothelial function is primarily used in scientific research, and to a lesser extent in clinical practice. Among the tools for assessing endothelial function, we can distinguish biochemical and physical methods, while physical methods can be divided into invasive and non-invasive methods. Flow-mediated dilation (FMD) is based on the ultrasound assessment of changes in the diameter of the brachial artery as a result of increased blood flow. FMD is a non-invasive, safe, and repeatable test, but it must be performed by qualified and experienced medical staff. The purpose of this paper is to present the literature review results on the assessment of endothelial function using the FMD method, including its methodology, applications in clinical practice and research, limitations, and future perspectives.