“Desirable difficulties” is a theory from cognitive science used to promote learning in a variety of contexts. The basic premise is that creating a cognitively challenging environment at the learning acquisition phase, by actively engaging learners in the retrieval of to-be-learned materials, promotes long-term retention. In this study, the degree of desirable difficulties was varied to identify how cognitively challenging the learning acquisition phase must be to benefit university-level students' learning of anatomy concepts. This is important to investigate as applied studies of desirable difficulties are less frequent than laboratory-based studies and the implementation of this principle may need to be tailored to the specific field of study, such as anatomy. As such, a read-read-read-read (R-R-R-R) condition was compared to read-generate-read-generate (R-G-R-G) and read-test-read-test (R-T-R-T) conditions. The three conditions varied in terms of how effortful the retrieval task was during the learning acquisition phase. R-R-R-R required little effort because participants passively read the materials four times. R-G-R-G required some effort to generate a response as participants completed a word fragment task during the learning acquisition phase. R-T-R-T was thought to be most demanding as participants performed a free recall task twice during the learning phase. With regard to the absolute amount of anatomy information recalled, the R-T-R-T condition was superior at both immediate and delayed (one week) assessment points. Thus, instructors and learners of anatomy would benefit from embedding more free recall components, or self-testing, into university-level course work or study practices.