Means and variances of the environmental thermal regime play an important role in determining the fitness of terrestrial ectotherms. Adaptive phenotypic responses induced by heterogeneous temperatures have been shown to be mediated by molecular pathways independent of the classic heat shock responses, however, an in-depth understanding of plasticity induced by fluctuating temperatures is still lacking. We investigated high and low temperature acclimation induced by fluctuating thermal regimes at two different mean temperatures, at two different amplitudes of fluctuation and across the developmental and adult life stages. For developmental acclimation, we found mildly detrimental effects of high amplitude fluctuations for critical thermal minima, while the critical thermal maxima showed a beneficial response to higher amplitude fluctuations. For adult acclimation involving shifts between fluctuating and constant regimes, cold tolerance was shown to be dictated by developmental temperature conditions irrespective of the adult treatments, while the acquired heat tolerance was readily lost when flies developed at fluctuating temperature were shifted to a constant regime as adults. Interestingly, we also found that effect of fluctuations at any life stage was gradually lost with prolonged adult maintenance suggesting a more prominent effect of fluctuations during developmental compared to adult acclimation in Drosophila melanogaster.