Abstract Introduction Clinical reasoning has been fostered with varying case formats including the use of virtual patients. Existing literature points to different conclusions regarding which format is most beneficial for learners with diverse levels of prior knowledge. We designed our study to better understand which case format affects clinical reasoning outcomes and cognitive load, dependent on medical students’ prior knowledge. Methods Overall, 142 medical students (3 rd to 6 th year) were randomly assigned to either a whole case or serial cue case format. Participants worked on eight virtual patients in their respective case format. Outcomes included diagnostic accuracy, knowledge, and cognitive load. Results We found no effect of case format on strategic knowledge scores pre- vs post-test (whole case learning gain = 3, 95% CI. -.01 to .01, serial cue learning gain = 3, 95% CI. -.06 to .00 p = .50). In both case formats, students with high baseline knowledge (determined by median split on the pre-test in conceptual knowledge) benefitted from learning with virtual patients (learning gain in strategic knowledge = 5, 95% CI .03 to .09, p = .01) while students with low prior knowledge did not (learning gain = 0, 95%CI −.02 to .02). We found no difference in diagnostic accuracy between experimental conditions (difference = .44, 95% CI −.96 to .08, p = .22), but diagnostic accuracy was higher for students with high prior knowledge compared to those with low prior knowledge (difference = .8, 95% CI 0.31 to 1.35, p < .01). Students with low prior knowledge experienced higher extraneous cognitive load than students with high prior knowledge (multiple measurements, p < .01). Conclusions The whole case and serial cue case formats alone did not affect students’ knowledge gain or diagnostic accuracy. Students with lower knowledge experienced increased cognitive load and appear to have learned less from their interaction with virtual patients. Cognitive load should be taken into account when attempting to help students learn clinical reasoning with virtual patients, especially for students with lower knowledge.