In this study we examined the effects of in vitro oxygenation and deoxygenation on the spun micro-haematocrit (packed cell volume or PCV) and the electronically measured mean cell volume (MCV) and haematocrit (Hct) of red blood cells. Because PCV and/or MCV and Hct are being used for calibration of haematology analysers, it is important that their potential variability caused by outside factors is known. We found that these parameters did vary with the oxygenation state of the erythrocyte and conclude that the effect is most likely caused by a combination of water shifts due to intracellular carbamate and bicarbonate formation, and conformational changes in haemoglobin. The results of the study have implications for whole blood calibration and for short term imprecision assessments of automated haematology analysers. To ensure consistent results, we recommend that blood specimens be fully oxygenated before reference work or reproducibility studies of MCV, Hct and/or PCV are attempted.