Understanding homeostasis is a goal of biology education curricula, as homeostasis is a core feature of living systems. Identifying and understanding the underlying molecular feedback mechanisms appear to be challenging for students. Understanding the properties and mechanisms of such complex homeostatic systems requires feedback loop reasoning, which is a part of systems thinking. Novices seem to struggle to 1) consider more than one initiating condition in cause-effect relationships and 2) track cause and effect across a sequence of processes. In this cross-sectional study, we analyzed how these factors impede feedback loop reasoning. High school and undergraduate students analyzed the organizational, behavioral, and modeling-related features of a homeostatic system (blood calcium regulation). Using multidimensional item response theory, we were able to confirm the three-dimensional structure of the theoretical systems-thinking model and to identify the factors causing item difficulty. As hypothesized, indirect relationships and derived inverse conditions are challenging factors for participants in the context of homeostasis across dimensions. Hence, we recommend paying special attention to these factors when teaching homeostasis as part of systems thinking. We assume that allowing students to reason from different initiating conditions in a learning setting may improve their systems-thinking skills.