Test-enhanced learning has successfully been used as a means to enhance learning and promote knowledge retention in students. We have examined whether this approach could be used in a biochemistry course to enhance student learning about lipids-related concepts. Students were provided access to two optional learning modules with questions related to the lipids block of the course. The vast majority of students (98.7-100%) used the optional modules. Student performance increased significantly with increased practice attempts (mean: first attempt--58.3%; high attempt--89.6%; p < 0.0001). This improvement was observed across all topics for both conceptual and structure recognition questions, and question types. A subset of questions were modified and included on formal assessments, and the results were compared to students that did not have access to modules (previous year). Incorporation of the modules resulted in a significant improvement in performance on the examination (year 1: 63.4%; year 2: 78.6%, p < 0.0001), including an increase in performance on questions requiring students to discriminate between highly similar topics. Importantly, comparison of student performance on quizzes and exams suggests that module use is associated with an increase in knowledge retention compared to restudy alone within the timeframe of the course. These results suggest that test-enhanced learning can be a valuable educational tool for development of metacognitive and test-taking skills that enhance student learning and understanding of course material.