The development of noninvasive analytical techniques is of interest to the field of chronobiology, in order to reveal the human metabolome that seems to show temporal patterns and to predict internal body time. We report on the real-time mass spectrometric analysis of human breath as a potential method to be used in this field. The breath of 12 subjects was analyzed during 9 days by secondary electrospray ionization-mass spectrometry (SESI-MS). The samples were collected during four time slots: morning (8:00-11:00), before lunch (11:00-13:00), after lunch (13:00-15:00), and late afternoon (15:00-18:00). A total of 203 mass spectra were statistically analyzed. Univariate analysis revealed a number of features with a marked temporal behavior. Principal component analysis/canonical analysis showed a clear temporal evolution of the breath patterns. A blind cross-validation yielded 84% of correct classifications of the time slot at which the breath samples were collected. We conclude that this approach seems to have potential for the investigation of biological clocks, including the description of internal body time, which may have important implications for the timing of pharmacotherapy.