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Abstract: Background: Isoprene in human breath is said to be related to cholesterol metabolism, and the possibility of the correlations with some clinical parameters has been studied. However, at this stage, no clear benefit of breath isoprene has been reported for clinical diagnosis. In this work, isoprene and acetone concentrations were measured in the breath of healthy and obese subjects using a needle-type extraction device for subsequent analysis in gas chromatography–mass spectrometry (GC-MS) to investigate the possibility of these compounds as an indicator of possible diseases. Methods: After measuring intraday and interday variations of isoprene and acetone concentrations in breath samples of healthy subjects, their concentrations were also determined in 80 healthy and 17 obese subjects. In addition, correlation between these breath concentrations and the blood tests result was studied for these healthy and obese subjects. Results: The results indicated successful determination of breath isoprene and acetone in this work, however, no clear correlation was observed between these measured values and the blood test results. Conclusions: Breath isoprene concentration may not be a useful indicator for obesity or hypercholesterolemia. [Copyright &y& Elsevier]