Misconceptions about physiology are a major threat to accurate scientific and clinical reasoning in medical education. Awareness is often mentioned as a prerequisite to achieve conceptual understanding; however, students are frequently unaware of their incorrect understanding. We explored the multitier approach as a tool to obtain insight into students' awareness and misconceptions regarding cardiovascular physiology. Biomedical sciences students (N = 81) participated in a diagnostic multitier assessment on cardiovascular physiology. Each question consisted of an answer tier and an explanation tier. Both tiers were paired with confidence tiers, i.e., 5-point Likert scales, which were used as an indicator for metacognitive evaluation, i.e., awareness. The average test score was 3.07 (maximum 4.0) for the answer tier only, and reduced to 1.57 when answer and explanation tiers were combined. A weak correlation (R2 = 0.13, P = 0.001) between students' confidence and their test scores was found for the combined responses. Correct combined answers were associated with an increase in confidence score of 0.27 vs. incorrect answers. Using a Bland-Altman analysis, we showed that students generally overestimated their knowledge. In total, 28.7% of all responses were classified as misconceptions, defined as incorrect answers paired with high confidence. In all, findings indicate that the multitier approach is useful to study students' conceptual understanding and uncover misconceptions on cardiovascular physiology. Furthermore, this study supports the need for metacognitive measures in order to improve teaching and learning in medical education.