In an undergraduate introductory biology laboratory course, we used a summative assessment to directly test the learning objective that students will be able to apply course material to increasingly novel and complex situations. Using a factorial framework, we developed multiple true-false questions to fall along axes of novelty and complexity, which resulted in four categories of questions: familiar content and low complexity (category A); novel content and low complexity (category B); familiar content and high complexity (category C); and novel content and high complexity (category D). On average, students scored more than 70% on all questions, indicating that the course largely met this learning objective. However, students scored highest on questions in category A, likely because they were most similar to course content, and lowest on questions in categories C and D. While we anticipated students would score equally on questions for which either novelty or complexity was altered (but not both), we observed that student scores in category C were lower than in category B. Furthermore, students performed equally poorly on all questions for which complexity was higher (categories C and D), even those containing familiar content, suggesting that application of course material to increasingly complex situations is particularly challenging to students.