Regular exercise reduces morbidity and mortality from cardiovascular diseases through cardiac and systemic adaptations including the increased coronary blood flow, an improved endothelial function, a slower progression of atherosclerotic processes as well as the stabilization of pre-existing lesions. However, long-term exercise is frequently associated with adverse cardiovascular events in 35-year older subjects because of a cardiac remodeling that predisposes to malignant arrhythmias and to an earlier atherosclerosis. Moreover, in athletes, ischemia is often silent with a higher risk of sudden cardiac death. The purpose of our observational study was to evaluate the efficacy of preparticipation screening in identifying the presence of coronary artery disease (CAD) in a population of asymptomatic master athletes. Between 2018 and 2019, 420 35-year older athletes (mean age: 55 + 17) underwent a preparticipation screening at the University of Pisa. None of them reported cardiovascular risk factors or previous cardiovascular diseases. After a physical examination, all subjects were submitted to spirometry and ECG stress test. The ECG stress test demonstrated signs of myocardial ischemia (ST depression > 1 mm) in 41 subjects; 3 athletes showed a frequency-dependent left bundle-branch block; 24 subjects had exercise-induced premature ventricular complexes during the ECG stress test. These subjects underwent further evaluations (Tc-99 myocardial perfusion SPECT and coronary angio-CT) to detect the presence of perfusion defects or coronary abnormalities: 26 athletes had an important coronary atherosclerosis with significant stenosis in one or more vessels and later subjected to Percutaneous transluminal coronary angioplasty (PTCA) or to coronary artery bypass grafting (CABG). 5 athletes presented a coronary myocardial bridge and 1 subject was demonstrated to have an anomalous origin of the left coronary. Preparticipation screening in 35-year-older athletes was able to detect the presence of obstructive coronary artery disease (CAD) in the 38% of suspected cases and anomalous origin or intramural coronary course in 8,8% of cases that showed electric abnormalities during the stress test. Despite recent conflicting opinions about the scientific value of the preparticipation screening in young athletes, the present study demonstrated that it was fundamental to early detect silent conditions that could predispose master athletes to adverse cardiovascular events.