The most prevalent type of ionizing radiation to which humans are exposed consists of mixed ionizing radiation fields. While exposure to a specific type of radiation (X and γ rays, α particles, among others) on cells and whole organisms has been widely studied, less is known about the biological effects following mixed ionizing radiation fields. This requires more information on the likelihood of subcellular effects, mainly DNA damage, according to the way energy is deposited within cells by ionizing particle tracks of different qualities. Due to their physical characteristics and the variety of nuclear atomic reactions, neutron exposures are of interest as they induce a mixed ionizing radiation field of secondary particles such as protons, alphas and electrons with very broad energy spectra. Therefore, we investigated, in situ, the evolution of DNA damage repair signaling, i.e. 53BP1 and γ-H2AX foci, in primary endothelial cells following complex irradiation with mixed radiation qualities by exposing cells to monoenergetic neutron fields of 2.5 or 15.1 MeV. The cells were placed on a human-mimicking water phantom positioned close to the source to maximize the absorbed dose. The assessment of the dose received by the cells was performed from primary neutrons fluence measurements and using Monte Carlo simulations with the Geant4 toolkit. The results shed light on the spatial rearrangement of a sub-set of foci 30 min post exposure, in the form of a linear pattern which occurred in 4 to 18% of cell nuclei depending on the neutrons energy and the irradiation configuration. These signatures were linked to realistic topologies of radiation-induced DNA damage at the cell population level, and their origin was analyzed by combining microdosimetric and nanodosimetric methods using the new MINAS TIRITH simulation tool which allowed its own validation. Our findings enabled us to study energy deposition and early cell damage from mixed radiation fields and obtain more accurate estimates of interaction probabilities that induce foci formation. This is crucial for assessing radiation therapy risk, where mixed radiation fields increase side effects and protecting space travelers from eventual health issues during long-duration space missions.