Galleria mellonella larvae are widely used for assessing the virulence of microbial pathogens and for measuring the in vivo activity of antimicrobial agents and produce results comparable to those that can be obtained using mammals. The aim of the work described here was to ascertain the effect of pre-incubation at 15°C for 1, 3, 6 or 10 weeks on the susceptibility of larvae to infection with Candida albicans and Staphylococcus aureus. Larvae infected with C. albicans after 1 week pre-incubation at 15°C showed 73.3 § 3.3% survival at 24 hours post-infection while those infected after 10 weeks pre-incubation showed 30 § 3.3% survival (P < 0.01). Larvae infected with S. aureus after 1 week preincubation showed 65.5 § 3.3% survival after 24 hours while those infected after 10 weeks pre-incubation showed 13.3 § 3.3% (P < 0.001). Analysis of the haemocyte density in larvae pre-incubated for 3–10 weeks showed a reduction in haemocytes over time but a proportionate increase in the density of granular haemocytes in the population as determined by FACS analysis. Proteomic analysis revealed decreased abundance of proteins associated with metabolic pathways (e.g. malate dehydrogenase, fructose-1,6-bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase) and prophenoloxidase. G. mellonella larvae are a useful in vivo model system but the duration of the pre-incubation stage significantly affects their susceptibility to microbial pathogens possibly as a result of altered metabolism.