Reference intervals (RIs) for haematology and serum biochemistry for donkeys in a temperate climate have been previously published using blood sample results from the resident population of a large donkey shelter in the UK. Periodic review of reference intervals is recommended to ensure their applicability to the patient population and changes in laboratory methods and technologies. The current study aimed to revise the previously published haematology and serum biochemistry values for the adult domestic donkey (Equus asinus) in the UK in the light of a change in analytical equipment at the Donkey Sanctuary laboratory, but also to refine the demography of the sample population with respect to age, physiology and clinical history. Clinical pathology results from 138 clinically healthy mature (4–24 years inclusive) female and castrated male donkeys selected from the resident population of the Donkey Sanctuary, were analysed retrospectively. The animals were blood sampled during the period February 2008 to June 2011 as part of a routine health screen prior to rehoming. Results for a total of 38 biochemical and haematological parameters were analysed including 3 previously unreferenced parameters in addition to those assessed in the previous study. The new reference intervals and median values show very poor transferability with recently derived reference intervals for non-Thoroughbred horses and only limited transferability with reference intervals previously published for donkeys in the UK. Of particular note is a marked reduction in the upper reference limit for triglycerides of 2.8 mmol/l (from 4.3 mmol/l) since this parameter is used to decide when donkeys are at risk of developing hyperlipaemia. This study demonstrates the value of intermittent review of reference intervals and refinement of study populations. Notwithstanding the caution with which reference interval data from different laboratories should be compared, the lack of transferability of results between donkeys and horses highlights the importance of use of species-appropriate reference intervals for clinical decision-making.