For the 2009 influenza A (H1N1) pandemic, vaccination and infection control were the main modes of prevention. A live attenuated H1N1 vaccine mimics natural infection and works by evoking a host immune response, but currently there are no easy methods to measure such a response. To determine if an immune response could be measured in exhaled breath, exhaled nitric oxide (FENO) and other exhaled breath volatiles using selected ion flow tube mass spectrometry (SIFT-MS) were measured before and daily for seven days after administering the H1N1 2009 monovalent live intranasal vaccine (FluMist (R), MedImmune LLC) in nine healthy healthcare workers (age 35 +/- 7 years; five females). On day 3 after H1N1 FluMist (R) administration there were increases in FENO (MEAN +/- SEM: day 0 15 +/- 3 ppb, day 3 19 +/- 3 ppb; p < 0.001) and breath isoprene (MEAN +/- SEM: day 0 59 +/- 15 ppb, day 3 99 +/- 17 ppb; p = 0.02). MS analysis identified the greatest number of changes in exhaled breath on day 3 with 137 product ion masses that changed from baseline. The exhaled breath changes on day 3 after H1N1 vaccination may reflect the underlying host immune response. However, further work to elucidate the sources of the exhaled breath changes is necessary.