Background and Purpose: Glucose tolerance tests (GTTs) are commonly used in preclinical studies but are poorly standardised. Male mice are often preferred due to more severe phenotypes which may aid in detecting drug effects. Using novel glucose telemetry in undisturbed mice, the effect of different pre-GTT protocols on blood glucose concentrations, GTTs and detection of drug effects were considered. Experimental Approach: Seven male and female C57Bl/6J mice (8-10 weeks) were implanted with HD-XG glucose telemetry devices. Mice were fasted for 16h overnight or 6h in the daytime following a whole cage change, cage change with retention of used bedding or no cage change prior to i.p.GTTs. Glucose tolerance following oral glucose gavage was compared to voluntary ingestion of gels. Using the most refined procedures, 250mg/kg oral metformin and 10nmol/kg i.p. exendin-4 were tested. Key Results: Blood glucose initially increased following cage changing at the start of the fast. For 6h fasting, retaining bedding reduced these initial responses and produced more timely glucose reductions whereas 16h fasts caused pronounced hypoglycaemia. Impaired glucose tolerance in males was exaggerated following 16h fasting or whole cage changes. Refined procedures including voluntarily ingested glucose gels blunted responses but the effects of exendin-4 and metformin were still observable in both sexes. Conclusion and Implications: Variations in GTT protocol can have profound effects on glucose homeostasis. Improved glucose tolerance due to protocol refinement and/or the use of females still allows for detection of drug effects providing evidence that more severe phenotypes are not required when testing drugs.