The objective of this study was assessment of the effect of aspiration flow, the nasal cycle, and time on nasal nitric oxide (nNO) concentrations in air sampled from one nostril during breathhold. nNO was measured in 45 healthy subjects (19 males, aged 18-45 years) from one nostril during breathholding. We compared nNO values and time to plateau in both nostrils with 3 aspiration flows (280, 700, 1,200 ml/min) and assessed the short-term and long-term reproducibility. Mean nNO values at flows of 280, 700 and 1,200 ml/min differed significantly (P < 0.01): 854, 474, 380 ppb, respectively. The (median) plateau was reached after 6, 4 and 3 s for the different flows. The within-subject coefficient of variability was always < 5%. We found no difference in nNO between left-, right-, largest or smallest nostril (P > 0.10). nNO values after 6, 24 h and 7 days were not significantly different from baseline (P > 0.10) and showed fair reproducibility. The highest aspiration flow was experienced as unpleasant. nNO can be measured in either nostril and shows no diurnal variation. The measurement is quick, reproducible, feasible and best accepted with an aspiration flow of 700 ml/min during breathhold for 10 s.