Temperature directly affects survival, development and reproduction in insects and thereby it is a key environmental driver for geographic distribution and population dynamics. This study aims at testing the survival of Drosophila melanogaster under constant low temperatures (CLTs) (2, 3, 4, and 5°C) vs. fluctuating thermal regimes (FTRs). In the latter, the cold stress period was interrupted daily by 2 h pulses at 20°C. Since acclimation enhances cold tolerance, we tested whether benefits of acclimation can combine with those of FTRs. Since D. melanogaster overwinters as non-reproductive adults, we tested if actively reproducing adults are more susceptible to cold stress than virgin females that have a much reduced reproductive activity. The results show that short interruptions of cold stress enhanced survival of adult flies. Survival was time- and temperature-dependent. Prior acclimation to low temperature allowed flies to better cope with cold stress under CLTs. On the other hand, acclimated flies did not profit from the benefits of FTRs and even showed lower survival under FTRs, probably because flies deacclimated during the periodic warm intervals. Gravid females were overall less cold tolerant than virgin females, and both survived better under FTRs. Cold survival at pupal stage was much lower than at adult stage, and no clear benefit of FTR was observed in this life stage. Our study highlights critical variables to take into account when designing experiments of prolonged exposure to low temperature in insects.