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Introduction Organisms use environmental cues to match their phenotype with the future availability of resources and environmental conditions. Changes in the magnitude and frequency of environmental cues such as photoperiod and temperature along latitudes can be used by organisms to predict seasonal changes. While the role of temperature variation on the induction of plastic and seasonal responses is well established, the importance of photoperiod for predicting seasonal changes is less explored. Materials and methods Here we studied changes in life-history and thermal stress resistance traits in Drosophila subobscura in response to variation in photoperiod (6:18, 12:12 and 18:6 light:dark cycles) mimicking seasonal variations in day length. The populations of D. subobscura were collected from five locations along a latitudinal gradient (from North Africa and Europe). These populations were exposed to different photoperiods for two generations, whereafter egg-to-adult viability, productivity, dry body weight, thermal tolerance, and starvation resistance were assessed. Results We found strong effects of photoperiod, origin of populations, and their interactions on life-history and stress resistance traits. Thermal resistance varied between the populations and the effect of photoperiod depended on the trait and the method applied for the assessment of thermal resistance. Perspectives Our results show a strong effect of the origin of population and photoperiod on a range of fitness-related traits and provide evidence for local adaptation to environmental cues (photoperiod by population interaction). The findings emphasize an important and often neglected role of photoperiod in studies on thermal resistance and suggest that cues induced by photoperiod may provide some buffer enabling populations to cope with a more variable and unpredictable future climate.