Selected ion flow tube-mass spectrometry (SIFT-MS) can measure volatile compounds in breath on-line in real time and has the potential to provide accurate breath tests for a number of inflammatory, infectious and metabolic diseases, including diabetes. Breath concentrations of acetone in type 2 diabetic subjects undertaking a long-term dietary modification programme were studied. Acetone concentrations in the breath of 38 subjects with type 2 diabetes were determined by SIFT-MS. Anthropomorphic measurements, dietary intake and medication use were recorded. Blood was analysed for beta hydroxybutyrate (a ketone body), HbA1c (glycated haemoglobin) and glucose using point-of-care capillary (fingerprick) testing. All subjects were able to undertake breath manoeuvres suitable for analysis. Breath acetone varied between 160 and 862 ppb (median 337 ppb) and was significantly higher in men (median 480 ppb versus 296 ppb, p = 0.01). In this cross-sectional study, no association was observed between breath acetone and either dietary macronutrients or point-of-care capillary blood tests. Breath analysis by SIFT-MS offers a rapid, reproducible and easily performed measurement of acetone concentration in ambulatory patients with type 2 diabetes. The high inter-individual variability in breath acetone concentration may limit its usefulness in cross-sectional studies. Breath acetone may nevertheless be useful for monitoring metabolic changes in longitudinal metabolic studies, in a variety of clinical and research settings.