The use of blood corticosterone and faecal corticosterone metabolites as biomarkers of post-surgical stress and pain in laboratory animals has increased during the last decade. However, many aspects of their reliability in laboratory mice remain uninvestigated. This study investigated serum corticosterone and adrenocorticotropic hormone (ACTH) in mice subjected to isoflurane anaesthesia and vasectomy, and mice subjected to isoflurane anaesthesia without surgery. Serum levels of corticosterone and ACTH after pre-treatment with dexamethasone were analysed to provide further information about the stress hormone profiles. Vasectomy resulted in an increase in corticosterone for at least four hours after surgery with a peak 30min after the mice regained righting reflex. Mice subjected to isoflurane anaesthesia without surgery had the highest level of serum corticosterone 5min after regained righting reflex and the level returned to baseline levels four hours after the procedure. In vasectomised mice, treated with dexamethasone, high levels of corticosterone remained 30min after the procedure, whereas the anaesthetised mice, treated with dexamethasone, had significantly lower levels of corticosterone compared to anaesthetised mice not treated with dexamethasone. Thus, dexamethasone effectively inhibited the corticosterone response in the anaesthetised-only mice, but not in the mice subjected to surgery. In conclusion, both isoflurane anaesthesia and vasectomy during isoflurane anaesthesia resulted in an increase in serum glucocorticoids, but the negative feedback mechanism of newly operated mice, was altered. This may have consequences for the interpretation of glucocorticoids measurements as a biomarker of post-surgical stress in mice.