The frequent exposure of terrestrial insects to temperature variation has led to the evolution of protective biochemical and physiological mechanisms, like the heat shock response which markedly increases the tolerance to heat stress. Insight into such mechanisms has, so far, mainly relied on selective studies of specific compounds or characteristics, or studies at the genomic or proteomic levels. In the present study we have used untargeted NMR metabolomic profiling to examine the biological response to heat stress in Drosophila melanogaster. The metabolite profile was analyzed during recovery after exposure to different thermal stress treatments and compared to untreated controls. Both moderate and severe heat stress gave clear effects on the metabolite profiles. The profiles clearly demonstrated that hardening by moderate heat stress led to a faster re-establishment of metabolite homeostasis after subsequent heat stress. Several metabolites were identified as responsive to heat stress, and could be related to known physiological and biochemical responses. The time-course of the recovery of metabolite homeostasis mirrored general changes in gene expression, showing that recovery follows the same temporal pattern at these two biological levels. Finally, our data show that heat-hardening permits a quicker return to homeostasis rather than a reduction of the acute metabolic perturbation, and that the re-establishment of homeostasis is important for obtaining maximal hardening effect. The results display the power of NMR metabolomic profiling for characterization of the instantaneous physiological condition, enabling direct visualization of the perturbation of and return to homeostasis.