Abstract Background Controversy remains as to whether exercise stress test (EST) is sufficient for risk evaluation in patients with preexcitation. Purpose This study aims to clarify the usefulness of EST in risk stratification in both asymptomatic and symptomatic patients, presenting with preexcitation. Methods This prospective study includes consecutive asymptomatic and symptomatic patients with preexcitation, referred for risk assessment. All participants performed an incremental EST (bicycle), prior to an electrophysiology study (EPS). Primary data from the EST included loss of preexcitation during exercise and from EPS included measurement of accessory pathway effective refractory period (APERP), shortest preexcited RR interval (SPERRI), and inducible arrythmia with use of a beta-adrenergic receptor agonist if deemed necessary. Results One hundred and sixty-four patients (59 asymptomatic, 105 symptomatic) completed an EST and EPS. Forty-five patients (27%) demonstrated low-risk findings on EST, of which 19 were asymptomatic and 26 were symptomatic. Six patients with low-risk EST findings had SPERRI/APERP≤250 ms at EPS, two of them were asymptomatic. The sensitivity, specificity, positive and negative predictive value (PPV, NPV) and accuracy of low-risk EST for excluding patients with SPERRI/APERP≤250 ms were 29%, 91%, 80%, 48%, and 55% respectively. The number of patients with inducible arrhythmia at EPS was similar in the asymptomatic 36 (69%) and in the symptomatic 73 (61%) group. Conclusions Sudden loss of preexcitation during EST has a low sensitivity and low NPV in excluding high-risk APs. EPS with use of isoproterenol should be considered to accurately assess the risk of patients with preexcitation regardless of symptoms.Figure