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Background Critical care glycaemic control protocols commonly have treatment adjustment (target) ranges spanning ≤2 mmol/L. These require precise point-of-care glucose measurement, unaffected by other variables, to avoid measurement errors increasing glycaemic variability and hypoglycaemic episodes (both strongly associated with mortality in critically ill patients). Methods A prospective 206 intensive care patient study was carried out. Arterial glucose concentrations were measured in duplicate using three point-of-care instruments (MediSense Precision PC χ, HemoCue DM and Radiometer 700), a central laboratory instrument (Siemens ADVIA), and in whole blood and plasma using the Yellow Springs Instruments 2300 instrument. Results Coefficients of variation for the MediSense, HemoCue, Radiometer and Siemens instruments were 5.1%, 2.5%, 2.1% and 2.3%, respectively. Compared with the Siemens instrument, the bias (95% limits of agreement) for the MediSense, HemoCue and Radiometer instruments were 0.0 (−1.4 to 1.4), 0.0 (−1.2 to 1.1) and −0.2 (−0.9 to 0.6) mmol/L, respectively. The whole blood–plasma glucose concentration difference was significantly affected by the haematocrit. MediSense and HemoCue instrument performances were substantially affected by haematocrit. MediSense instrument performance was also affected by pH and PaO2. Radiometer instrument performance was not affected by haematocrit, pH or PaO2. Conclusions The MediSense instrument was too imprecise for use in critically ill patients. The haematocrit range seen is too great to allow fixed-factor conversion between whole blood and plasma values, substantially affecting the accuracy of both glucose meters. However, the Radiometer instrument was unaffected by the haematocrit, pH or pO2, resulting in a performance equivalent to the laboratory method. Instrument performance differences may therefore partially explain the differing results of tight glycaemic control therapy trials.