There is interest in pinpointing genes and physiological mechanisms explaining intra- and interspecific variations in cold tolerance, because thermal tolerance phenotypes strongly impact the distribution and abundance of wild animals. Laboratory studies have highlighted that the capacity to preserve water and ion homeostasis is linked to low temperature survival in insects. It remains unknown, however, whether adaptive seasonal acclimatization in free-ranging insects is governed by the same physiological mechanisms. Here, we test whether cold tolerance in field-caught Drosophila subobscura is high in early spring and lower during summer and whether this transition is associated with seasonal changes in the capacity of flies to preserve water and ion balance during cold stress. Indeed, flies caught during summer were less cold tolerant, and exposure of these flies to sub-zero temperatures caused a loss of haemolymph water and increased the concentration of K+ in the haemolymph (as in laboratory-reared insects). This pattern of ion and water balance disruption was not observed in more cold-tolerant flies caught in early spring. Thus, we here provide a field verification of hypotheses based on laboratory studies and conclude that the ability to maintain ion homeostasis is important for the ability of free-ranging insects to cope with chilling.