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This study investigated whether simulated darkness influences the affective appraisal of a desktop virtual environment (VE). In the real world darkness often evokes thoughts of vulnerability, threat, and danger, and may automatically precipitate emotional responses consonant with those thoughts (fear of darkness). This influences the affective appraisal of a given environment after dark and the way humans behave in that environment in conditions of low lighting. Desktop VEs are increasingly deployed to study the effects of environmental qualities and (architectural or lighting) interventions on human behaviour and feelings of safety. Their (ecological) validity for these purposes depends critically on their ability to correctly address the user’s cognitive and affective experience. However, it is currently not known how and to what extent simulated darkness in desktop (i.e., non-immersive) VEs affects the user’s affective appraisal of the represented environment. In this study young female volunteers explored either a daytime or a night-time version of a desktop VE representing a deserted prototypical Dutch polder landscape. The affective appraisal of the VE and the emotional response of the participants were measured through self-report. To enhance the personal relevance of the simulation, a fraction of the participants was led to believe that the virtual exploration tour would prepare them for a follow-up tour through the real world counterpart of the VE. The results show that the VE was appraised as slightly less pleasant and more arousing in simulated darkness (compared to a daylight) condition. The fictitious follow-up assignment had no emotional effects and did not influence the affective appraisal of the VE. Further research is required to assess on the validity of desktop VEs for both etiological (e.g., the effects of signs of darkness on navigation behaviour and fear of crime) and intervention (e.g., effects of street lighting on feelings of safety) research.