ECG is a simple, quick, cheap and non-invasive diagnostic technique [48], that provides a graphic recording of the electrical cardiac activity. It is nowadays widely used for CV screening, given its important role in reducing SCD rate [49], but its cost effectiveness, the need for experienced physicians to correctly interpret it and a high false positive rate are criticisms often moved about it [12, 50, 51]. ECG changes in athletes are common and usually reflect adaptive structural and electrical remodeling of the heart in response to regular training [52, 53, 54, 55, 56]. Furthermore, ECG adaptations may vary according to demographic characteristics, such as age, sex and ethnicity, as well as the type of sport and level of training. Based on the International Recommendations for ECG interpretation in athletes, which should be applied only to those exercising vigorously for at least 4–8 hours per week [44], ECG findings in athletes are classified as normal, abnormal and borderline (Table 1, Ref. [44]): if one abnormal or two borderline findings together are detected, further evaluation must be performed. However, some of these adaptive changes overlap with patterns reflective of underlying pathology. Accurate interpretation of the ECG in asymptomatic athletes is of paramount importance to avoid unnecessary further investigations (given the possibility of false positive findings of this technique [50, 51]) or sport disqualification, and prevent serious consequences, including SCD, in case of high-risk cardiovascular conditions. A proposal of a modified algorithm for ECG interpretation in children athletes has been recently hypothesized [57].

Due to its ability to provide information on cardiac morphology, function and hemodynamics, its low cost and wide and easy availability, the use of echocardiography in athlete’s evaluation is increasing [58, 65, 66, 67], also given that the low acoustic chest impedance of the athletic population makes it possible to obtain high-quality images [16].

Cardiovascular adaptations of an athlete’s heart include balanced increases in all heart chambers. While interest has largely focused on the LV in the past, attention has recently been directed to other structures such as the RV, the atria, and the aorta [68, 69]: adaptations to physical activity include a proportional increase in the left and right cardiac cavity sizes, increased LV wall thickness and LV mass, and supra-normal indices of systolic and diastolic function [70, 71, 72, 73] (Fig. 2). These adaptations, strictly dependent upon the duration, type and intensity of training, are often benign and physiological but may sometimes predispose to pathological conditions [74].

Several reference values about age, gender, ethnicity, and sports disciplines have been published in the literature by different study groups (Table 2, Ref. [75, 76, 77, 78, 79, 80]; Table 3, Ref. [80, 81]). However, we currently lack universally accepted cut-offs for basic echocardiographic measurements [75, 82], and therefore there are no unanimous recommendations about the use of echocardiographic cut-offs to distinguish between physiological and pathological adaptations. Indeed, comprehensive nomograms including sufficient sample size (of both genders), evaluating different ages (including master athletes) and ethnicities and various sports, evaluating a complete dataset of 2D (and new 3D and strain analysis indexes) echocardiographic measures, and built using a rigorous statistical approach (uniform normalization and way to express normalized data—preferably as Z-scores) are still missing in current literature [83]. Therefore, care is needed when interpreting this exam.

While a mild dilatation of the aorta can sometimes be observed, particularly in some categories of athletes (i.e., master endurance athletes), a more-than-mild dilatation is not part of the athlete’s heart and should warrant further investigations in case of an aortic root more than 40 mm in males (an indexed values of 20 mm/m${}^2$ for allometric scale) and 34 mm in females and more than 34 mm for the proximal ascending aorta [65, 73].

In the last decades, advances in ultrasound technology have evolved echocardiography from simple M-mode to 2-dimensional imaging, Doppler assessments, 3-dimensional (3D) anatomical imaging, and dimensional analysis of myocardial deformation [84]. Speckle-tracking echocardiography is very useful in evaluating the athletes’ heart [85], especially in identifying regional wall motion abnormalities and pre-clinical impairment in HCM and DCM. The mean value of global longitudinal strain in athletes is –18.1 $\pm$ 2.2% in LV and –27 $\pm$ 6% in RV [65] (Fig. 3). Three-dimensional echocardiography has added quantitative information to assess the athlete’s heart; cardiac volumes and mass can be estimated more precisely than 2D echo without geometric assumptions [86]. Moreover, myocardial work, calculated by adjusting myocardial deformation to the instantaneous LV pressure, has been recently proposed to have a role in the athlete’s heart diagnostic process, due to its less dependency on loading contraction than global longitudinal strain [87].

EST is the most widely available functional test. The continuous ECG and blood pressure (BP) monitoring of the subject during a treadmill or cycle-ergometer incremental test, provides information on exercise capacity, heart rate and BP response to exercise, other than exercise-induced abnormalities, including arrhythmias [88]. It can be used for diagnostic, prognostic, or functional evaluation purposes [89], and it can also be adopted using different protocols based, for instance, on the type of sports practiced by the athlete [65]. Indeed, both treadmill and cycle ergometer protocols have their strengths and disadvantages and should be used with a precise aim in athletes [90]. However, some contraindications to EST must be considered [91]. In addition, EST requires specific care because of the wide range of normal findings, the use of different stress-inducing protocols, and the lack of generally accepted reference values [92].

In athletes over 35 years of age, EST investigates the presence of a silent ischemic cardiovascular disease through specific alterations in ST-segment and T-wave. However, it is less specific for myocardial ischemia than other functional tests, especially in asymptomatic and low-risk individuals [8]: for example, an asymptomatic upsloping ST-segment depression with normalization in the early (1 min) phase of recovery should not be considered pathological [93].

EST also permits assessing BP changes during exercise. An exaggerated BP response to exercise should lead to starting or optimizing antihypertensive medical therapy and performing a cardiologic evaluation, even if the athlete is normotensive at rest since it predicts incident and early hypertension in athletes [94]. In a large cohort of elite athletes undergoing EST, the 95th percentile of BP values was 220/85 mmHg in males and 200/80 mmHg in females [95]. Also, a decrease in BP during the test is not normal and should be further investigated.

Even if ventricular arrhythmias may be unrelated to heart diseases, some of them could be a marker for an arrhythmogenic condition in athletes with no relevant history, normal physical examination, and resting ECG, and therefore EST plays a pivotal role in describing its effort-related characteristics [96]. Other arrhythmias that can be studied through EST are atrial fibrillation, first- or second-degree AV block, and asymptomatic pre-excitation [65]. Also, QT interval adaptation to exercise and recovery phase is an important phenomenon to consider when evaluating an EST.

ECG continuous monitoring is a method that provides more information for the detection of cardiac rhythm alterations than resting 12-lead ECG recording. ECG Holter monitoring recording with 12-leads configuration should always be preferred to determine the origin of ventricular arrhythmias (morphology and axis) and the presence of ischemia [97]. The monitoring period is usually 24 hours, even if it may be longer in specific cases, and should always include a training session, to reproduce as much as possible the “natural” physical effort of the athlete: this allows to study the response of the arrhythmias to exercise and to elicit arrhythmias that are in relation with the effort. However, ECG Holter monitoring in athletes is often rich in motion artifacts; therefore, an experienced physician is required to interpret it.

Life-threatening arrhythmias are infrequent among young athletes who require ECG monitoring, whereas their presence may suggest an underlying cardiac disease according to some specific characteristics [98] (Table 4, Ref. [96, 97]). A diary in which the patient reports the main daily activities (i.e., exercise sessions, sleeping times, etc.), any drug therapy taken or symptoms experienced should always be part of the assessment.

Progress in science and technology has led to the development of numerous devices, such as the external loop recorder, event recorders or wearables, for assessing cardiac arrhythmias, that are nowadays available for patients and should be used in selected cases (i.e., symptomatic athletes with infrequent symptoms) and with careful interpretation [99, 100].

CPET is a valuable tool to evaluate the responses of the cardiac, pulmonary, vascular, and musculoskeletal systems to exercise [101, 102, 103, 104]. Although still underutilized, its high reproducibility offers important prognostic and diagnostic information [105] and can be integrated with other imaging techniques [106]. Different from an EST, CPET involves measurements of respiratory oxygen uptake, carbon dioxide production, and ventilatory measures during a symptom-limited exercise test. CPET indications in athletes are manifold, including cardiorespiratory fitness estimation, evaluation of symptoms of unexplained origin and exercise prescription [107]. It is known that highly trained athletes have higher cardiorespiratory fitness compared to untrained individuals or low-trained athletes [108]. Therefore, cardiorespiratory fitness considered in the normal predicted ranges may mask latent disorders or physiological impairments in athletes. For this reason, interpreting CPET results requires caution within the clinical context, as predicted gas exchange parameters have been derived in the general population [109]. Moreover, athletes show further differences in exercise hemodynamic response and gas exchange parameters compared to non-athletes, including higher cardiac output, faster heart rate recovery, higher prevalence of exercise-induced arterial hypoxemia, and lower breathing reserve [107, 110] (Table 5, Ref. [107, 110, 111, 112]), even if reference values have yet to be determined [113]. Knowing these parameters in the context of the athlete’s physiological response to exercise could help guide the differential diagnosis between the athlete’s heart and underlying CV diseases [114].

In clinical settings, CPET is generally used to evaluate the etiology of unexplained symptoms such as exertional dyspnea, chest discomfort, and fatigue. Moreover, excessive training load without an adequate recovery period exposes athletes to decreased performance and sometimes even to overtraining syndrome. CPET may be particularly useful in this condition.

Finally, CPET can be used for a tailored exercise prescription, not only to improve the performance of elite endurance athletes but also in patients at risk of and with CV disease, especially those who are older and engage for the first time in moderate to vigorous physical activity [111, 115]. Through the identification of ventilatory thresholds, the physician may draw out a personally tailored program with the appropriate level of intensity associated with possible enhancements for healthy athletes and proven benefits for patients with chronic diseases [116, 117]. Moreover, CPET should be part of the routine assessment of patients with cardiomyopathies who wish to exercise to obtain information about functional capacity and risk stratification [97, 106, 118, 119].

ESE is a reliable, safe, non-invasive imaging test that provides a dynamic cardiac function evaluation. Combined with clinical and ECG data, ESE helps detect cardiac abnormalities that may not occur at rest, such as exercise-induced ischemia in athletes with suspected coronary artery disease or congenital coronary artery anomalies [121]. Furthermore, ESE can assess contractile reserve during exercise in endurance athletes with LV and/or RV dilatation and mildly reduced ejection fraction at rest: an increase of LV EF of at least 15% during exercise may support the diagnosis of athlete’s heart [122]. Finally, ESE may be useful in athletes with valvular heart disease, providing information about exercise tolerance, biventricular contractile reserve, changes in hemodynamics (LV filling pressure, pulmonary pressure), and valvular functional parameters (transvalvular gradients, regurgitation entity—i.e., bicuspid aortic valve) [78].

Pharmacological stress is generally not indicated in athletes and an exercise test is usually performed through a bed cycle ergometer. Since the time for image acquisition is limited, the echocardiographic protocol is usually tailored to the clinical indication [65]. However, even if some limitations to the use of ESE exist, improvements in imaging equipment and technology, and allowing the movement towards more robust quantitative analysis, have led ESE to become a valuable tool in the diagnostic process of athlete’s heart.

CMR is an established imaging modality for the cardiovascular assessment of athletes. It is a third-tier diagnostic tool that helps to discriminate between physiology and pathology [65], and it is superior to echocardiography in differentiating athlete’s heart from structural and functional change [123]. The limitations of CMR include, among others, high cost, limited accessibility and claustrophobia, other than untested or low interobserver variability [124].

CMR is the gold standard for defining biventricular volumes and mass, and quantification of volumes and flow (Table 7, Ref. [125]) [126], providing advanced myocardial tissue characterization with excellent accuracy and precision. CMR has the incremental benefit of allowing tissue characterization by identifying myocardial inflammation and fat infiltration through T1 and T2 weighted images and mapping. CMR allows the detection of replacement fibrosis by late gadolinium enhancement (LGE) imaging, also pointing to the description of ischemic vs nonischemic patterns of myocardial damage [127]. As such, CMR supports the diagnosis of myocarditis and cardiomyopathies [128], such as HCM [129] and ACM [130, 131, 132, 133]. To differentiate between pathologic modification and physiologic remodeling, cardiac volumes and masses should always be compared to reference ranges deriving from CMR studies on healthy athletes [134], and adjusted to several factors, including type of sport, static and dynamic component, training hours per week, body surface area, age, gender, and ethnicity [135] (Fig. 4).

In the last years, it is spreading the use of stress CMR that has the advantages to assess biventricular function, wall motion and valve function during exercise, even if it requires high levels of training, dedicated devices, and long-scan times [136].

CCT shows high accuracy in evaluating coronary atherosclerosis and coronary origin and course [121]. The assessment of coronary arteries by CCT is non-invasively performed and requires a very low radiation dose, thanks to the latest generation scanners (from 0.7 to 1 mSv with optimized acquisition parameters and protocols [136]). Therefore, depending on the local availability and expertise, CCT may be considered in athletes with symptoms suggestive of CAD and in older, asymptomatic athletes with risk factors for CV disease or equivocal exercise stress test. Indeed, it has been recently theorized the use of CCT in the screening process of mature athletes increases the negative predictive value for excluding coronary artery disease [137]. Moreover, CCT should be considered when a precise definition of proximal coronary anatomy or characterization of great vessel morphology is indicated [136]. When dilatation of the aortic root or ascending aorta is suspected, at least one comprehensive aortic tomographic assessment by CT angiography or angio-MR should be performed [73]. Cardiac CT well visualizes pericardial thickening and calcification, and CT attenuation values may differentiate pericardial fluid contents. Once anatomical abnormalities have been detected, a CV functional assessment performed during exercise is required to evaluate their functional clinical impact.

However, due to ionizing radiation exposure and high costs, this imaging modality is not recommended as the first-line technique for young athletes.

Myocardial perfusion scintigraphy techniques are generally considered a valuable diagnostic and prognostic modality and often used for further diagnostic evaluation in athletes with electrocardiographic findings indicative of myocardial ischemia in the PPS [138]. SPECT or PET can research exercise-induced ischemia and stratify the risk of athletes with suspected or known CAD, anomalous origin, or course of coronary arteries (e.g., myocardial bridging) [139]. The accuracy of both PET and SPECT in detecting CAD is excellent [140]. However, PET may be preferred in balanced 3-vessel disease since it permits absolute quantification of myocardial blood flow. Conversely, SPECT can only provide semi-quantitative values (normalized to the maximum value), failing to detect relative perfusion differences [141, 142]. Moreover, even SPECT specificity in competitive athletes has to be considered reduced, given that myocardial perfusion defects can be present also in healthy young male athletes, and they are associated with LV hypertrophy and no wall motion abnormalities on echocardiography [142]. Thus, cardiac nuclear imaging in the athlete’s setting is more suitable for research purposes than for a clinical application and should not be recommended as a first-line test in competitive athletes [123]. According to European guidelines [8], nuclear imaging may also be considered an alternative or complementary exam to ESE or CCT for evaluating asymptomatic individuals aged $>$35 years with CV risk factors before engaging in high or very high-intensity sports.

Genetic testing is a valuable tool for diagnosing several inherited cardiac disorders [120, 143]. In athletes, it can be beneficial in terms of diagnosis, management, decisions relating to sports participation, and prognosis [144]. Moreover, identifying disease-causing mutations allows cascade screening in first-degree family members due to the autosomal dominant pattern in most inherited cardiac disorders [145].

The diagnostic yield of genetic testing is significantly different according to the clinical phenotype of the athlete. From a general point of view, genetic testing in diagnosing an inherited cardiac disorder is useful in individuals with clear phenotypes. In recent years, attention has been given to the diagnostic role of genetic testing in an individual who exhibits an overlapping phenotype between inherited cardiac disease and athlete’s heart [146, 147]. In selected cases, when a comprehensive clinical evaluation is suspicious but fails to reach a definitive diagnosis of inherited cardiac disease, genetic testing may be considered, keeping in mind the specific diagnostic yield for each disease and that it can be even lower in athletes. The benefit of genetic testing should always be weighed with potential harm. The genetic testing panel should only include genes with supporting solid evidence to cause the athletes’ clinical phenotype to minimize the identification of a variant of uncertain significance or allelic variants associated with the different clinical phenotype [148], which increases the difficulties inherent to the interpretation of genetic testing results.

Since the diagnostic yield of genetic testing is significantly different according to the clinical phenotype of the athlete, physicians involved in the athlete’s management should have a solid understanding of the indications, strengths and limitations of genetic testing (Table 8, Ref. [120]).