Multiple-choice assessments provide a straightforward way for instructors of large classes to collect data related to student understanding of key concepts at the beginning and end of a course. By tracking student performance over time, instructors receive formative feedback about their teaching and can assess the impact of instructional changes. The evidence of instructional effectiveness can in turn inform future instruction, and vice versa. In this study, we analyzed student responses on an optimized pretest and posttest administered during four different quarters in a large-enrollment biochemistry course. Student performance and the effect of instructional interventions related to three fundamental concepts--hydrogen bonding, bond energy, and pK[subscript a]--were analyzed. After instructional interventions, a larger proportion of students demonstrated knowledge of these concepts compared with data collected before instructional interventions. Student responses trended from inconsistent to consistent and from incorrect to correct. The instructional effect was particularly remarkable for the later three quarters related to hydrogen bonding and bond energy. This study supports the use of multiple-choice instruments to assess the effectiveness of instructional interventions, especially in large classes, by providing instructors with quick and reliable feedback on student knowledge of each specific fundamental concept.