Background: Standardized patients (SPs) have been one of the popular assessment methods in clinical teaching for decades, although they are resource intensive. Nowadays, simulated virtual patients (VPs) are increasingly used because they are permanently available and fully scalable to a large audience. However, empirical studies comparing the differential effects of these assessment methods are lacking. Similarly, the relationships between key variables associated with diagnostic competences (ie, diagnostic accuracy and evidence generation) in these assessment methods still require further research. Objective: The aim of this study is to compare perceived authenticity, cognitive load, and diagnostic competences in performance-based assessment using SPs and VPs. This study also aims to examine the relationships of perceived authenticity, cognitive load, and quality of evidence generation with diagnostic accuracy. Methods: We conducted an experimental study with 86 medical students (mean 26.03 years, SD 4.71) focusing on history taking in dyspnea cases. Participants solved three cases with SPs and three cases with VPs in this repeated measures study. After each case, students provided a diagnosis and rated perceived authenticity and cognitive load. The provided diagnosis was scored in terms of diagnostic accuracy; the questions asked by the medical students were rated with respect to their quality of evidence generation. In addition to regular null hypothesis testing, this study used equivalence testing to investigate the absence of meaningful effects. Results: Perceived authenticity (1-tailed t81=11.12; P<.001) was higher for SPs than for VPs. The correlation between diagnostic accuracy and perceived authenticity was very small (r=0.05) and neither equivalent (P=.09) nor statistically significant (P=.32). Cognitive load was equivalent in both assessment methods (t82=2.81; P=.003). Intrinsic cognitive load (1-tailed r=−0.30; P=.003) and extraneous load (1-tailed r=−0.29; P=.003) correlated negatively with the combined score for diagnostic accuracy. The quality of evidence generation was positively related to diagnostic accuracy for VPs (1-tailed r=0.38; P<.001); this finding did not hold for SPs (1-tailed r=0.05; P=.32). Comparing both assessment methods with each other, diagnostic accuracy was higher for SPs than for VPs (2-tailed t85=2.49; P=.01). Conclusions: The results on perceived authenticity demonstrate that learners experience SPs as more authentic than VPs. As higher amounts of intrinsic and extraneous cognitive loads are detrimental to performance, both types of cognitive load must be monitored and manipulated systematically in the assessment. Diagnostic accuracy was higher for SPs than for VPs, which could potentially negatively affect students’ grades with VPs. We identify and discuss possible reasons for this performance difference between both assessment methods.