The analysis of exhaled metabolites has become a promising field of research in recent decades. Several volatile organic compounds reflecting metabolic disturbance and nutrition status have even been reported. These are particularly important for long-term measurements, as needed in medical research for detection of disease progression and therapeutic efficacy. In this context, it has become urgent to investigate the effect of fasting and glucose treatment for breath analysis. In the present study, we used a model of ventilated rats that fasted for 12 h prior to the experiment. Ten rats per group were randomly assigned for continuous intravenous infusion without glucose or an infusion including 25 mg glucose per 100 g per hour during an observation period of 12 h. Exhaled gas was analysed using multicapillary column ion-mobility spectrometry. Analytes were identified by the BS-MCC/IMS database (version 1209; B & S Analytik, Dortmund, Germany). Glucose infusion led to a significant increase in blood glucose levels (p < 0.05 at 4 h and thereafter) and cardiac output (p < 0.05 at 4 h and thereafter). During the observation period, 39 peaks were found collectively. There were significant differences between groups in the concentration of ten volatile organic compounds: p < 0.001 at 4 h and thereafter for isoprene, cyclohexanone, acetone, p-cymol, 2-hexanone, phenylacetylene, and one unknown compound, and p < 0.001 at 8 h and thereafter for 1-pentanol, 1-propanol, and 2-heptanol. Our results indicate that for long-term measurement, fasting and the withholding of glucose could contribute to changes of volatile metabolites in exhaled air.