A flipped classroom is a growing pedagogy in higher education. Many research studies on the flipped classroom have focused on student outcomes, with the results being positive or inconclusive. A few studies have analyzed confounding variables, such as student's previous achievement, or the impact of a flipped classroom on long-term retention and knowledge transfer. In the present study, students in a Doctor of Physical Therapy program in a traditional style lecture of gross anatomy (n = 105) were compared to similar students in a flipped classroom (n = 112). Overall, students in the flipped anatomy classroom had an increase in semester average grades (P = 0.01) and performance on higher-level analytical questions (P < 0.001). Long-term retention and knowledge transfer was analyzed in a subsequent semester's sequenced kinesiology course, with students from the flipped anatomy classroom performing at a higher level in kinesiology (P < 0.05). Student's pre-matriculation grade point average was also considered. Previously lower performing students, when in a flipped anatomy class, outperformed their traditional anatomy class counterparts in anatomy semester grades (P < 0.05), accuracy on higher-level analytical anatomy multiple-choice questions (P < 0.05) and performance in subsequent course of kinesiology (P < 0.05). This study suggests that the flipped classroom may benefit lower performing student's knowledge acquisition and transfer to a greater degree than higher performing students. Future studies should explore the underlying reasons for improvement in lower performing students.