An understanding of the structural properties that allow for optimal cathode performance, and their origin, is necessary for devising advanced cathode design strategies and accelerating the commercialisation of next-generation cathodes. High-voltage, Fe- and Mg-substituted LiNi0.5Mn1.5O4 cathodes offer a low-cost and cobalt-free, yet energy-dense alternative to commercial cathodes. In this work, we explore the effect of substituents on several important structure properties including Ni/Mn ordering, charge distribution and extrinsic defects. In the cation-disordered samples studied, we observe a correlation between increased Fe/Mg substitution, Li-site defects and Li-rich impurity phase formation - the concentrations of which are greater for Mg-substituted samples. We attribute this to the lower formation energy of MgLi defects when compared to FeLi defects. Li-site defect-induced impurity phases consequently alter the charge distribution of the system, resulting in increased [Mn3+] with Fe/Mg substitution. In addition to impurity phases, other charge compensators were also investigated to explain the origin of Mn3+ (extrinsic defects, [Ni3+], oxygen vacancies and intrinsic off-stoichiometry), although their effects were found to be negligible. This article is protected by copyright. All rights reserved.