Background: Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we aimed to develop mathematical models to explore theoretical cellular states underpinning the empirically observed responses of macrophages following lipopolysaccharide (LPS) challenge. Methods: We obtained empirical data following primary and secondary responses to LPS in two in vitro cellular models (bone marrow-derived macrophages or BMDMs, and RAW 264.7 cells) and single-cell protein measurements for four key inflammatory mediators: TNF, IL-6, pro-IL-1β, and NOS2, and used mathematical modelling to understand heterogeneity. Results: For these four factors, we showed that macrophage community AIH is dependent on LPS dose and that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical three-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a four-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS. Conclusions: Overall, we provide a mathematical model for studying macrophage ecology and community dynamics that can be used to elucidate the role of phenotypically negative macrophage populations in AIH and, primary and secondary responses to LPS.