Human breath contains an abundance of volatile organic compounds (VOCs). Analysis of breath VOC may be used for diagnosis of various diseases or for on-line monitoring in anesthesia and intensive care. However, VOC concentrations largely depend on the breath sampling method and have a large inter-individual variability. For the development of breath tests, the influence of breath sampling methods and study subject characteristics on VOC concentrations has to be known. Therefore, we investigated the VOC isoprene in 62 study subjects during anesthesia and 16 spontaneously breathing healthy volunteers to determine (a) the influence of artificial and spontaneous ventilation and (b) the influence of study subject characteristics on breath isoprene concentrations. We used ion molecule reaction mass spectrometry for high-resolution breath-by-breath analysis of isoprene. We found that persons during anesthesia had significantly increased inspiratory and end-expiratory isoprene breath concentrations. Measured isoprene concentrations (median [first quartile-third quartile]) were in the anesthesia group: 54 [40-79] ppb (inspiratory) and 224 [171-309] ppb (end-expiratory), volunteer group: 14 [11-17] ppb (inspiratory) and 174 [124-202] ppb (end-expiratory). Higher end-tidal CO(2) concentrations in ventilated subjects were associated with higher expiratory isoprene levels. Furthermore, inspiratory and end-expiratory isoprene concentrations were correlated during anesthesia (r = 0.603, p < 0.001). Multivariate analysis showed that men had significantly higher end-expiratory isoprene concentrations than women. Rebreathing of isoprene from the anesthesia machine possibly accounts for the observed increase in isoprene in the anesthesia group.