Herpes simplex virus-1 (HSV-1) establishes a latent infection in peripheral neurons and periodically reactivates in response to a stimulus to permit transmission. In vitro models using primary neurons are invaluable to studying latent infection because they use bona fide neurons that have undergone differentiation and maturation in vivo. However, culture conditions in vitro should remain as close to those in vivo as possible. This is especially important when considering minimizing cell stress, as it is a well-known trigger of HSV reactivation. We recently developed an HSV-1 model system that requires neurons to be cultured for extended lengths of time. Therefore, we sought to refine culture conditions to optimize neuronal health and minimize secondary effects on latency and reactivation. Here, we demonstrate that culturing primary neurons under conditions closer to physiological oxygen concentrations (5% oxygen) results in cultures with features consistent with reduced stress. Furthermore, culture in these lower oxygen conditions diminishes the progression to full HSV-1 reactivation despite minimal impacts on latency establishment and earlier stages of HSV-1 reactivation. We anticipate that our findings will be useful for the broader microbiology community as they highlight the importance of considering physiological oxygen concentration in studying host-pathogen interactions.IMPORTANCEEstablishing models to investigate host-pathogen interactions requires mimicking physiological conditions as closely as possible. One consideration is the oxygen concentration used for in vitro tissue culture experiments. Standard incubators do not regulate oxygen levels, exposing cells to oxygen concentrations of approximately 18%. However, cells within the body are exposed to much lower oxygen concentrations, with physiological oxygen concentrations in the brain being 0.55%-8% oxygen. Here, we describe a model for herpes simplex virus 1 (HSV-1) latent infection using neurons cultured in 5% oxygen. We show that culturing neurons in more physiological oxygen concentrations improves neuronal health to permit long-term studies of virus-cell interactions and the impact on reactivation.