Rodent behavior is affected by different environmental conditions. These do not only comprise experimental and housing conditions but also familiarization with the experimenter. However, specific effects on pain-related behavior and chronic pain conditions have not been examined. Therefore, we aimed to investigate the impact of different housing conditions, using individually ventilated and standard open top cages, inverted day-night cycles, and experimenter familiarization on male mice following peripheral neuropathy using the spared nerve injury (SNI) model. Using a multimodal approach, we evaluated evoked pain-related- using von Frey hair filaments, measured gait pattern with the CatWalk system, assessed anxiety- and depression-like behavior with the Elevated plus maze and tail suspension test, measured corticosterone metabolite levels in feces and utilized an integrative approach for relative-severity-assessment. Mechanical sensitivity differed between the cage systems and experimenter familiarization and was affected in both sham and SNI mice. Experimenter familiarization and an inverted day-night cycle reduced mechanical hypersensitivity in SNI and sham mice. SNI mice of the inverted day-night group displayed the slightest pronounced alterations in gait pattern in the Catwalk test. Anxiety-related behavior was only found in SNI mice of experimenter-familiarized mice compared to the sham controls. In addition, familiarization reduced the stress level measured by fecal corticosteroid metabolites caused by the pain and the behavioral tests. Although no environmental condition significantly modulated the severity in SNI mice, it influenced pain-affected phenotypes and is, therefore, crucial for designing and interpreting preclinical pain studies. Moreover, environmental conditions should be considered more in the reporting guidelines, described in more detail, and discussed as a potential influence on pain phenotypes.