The collection of exhaled breath condensate (EBC) is simple and non-invasive, however, there are few data on the methodological aspects affecting concentrations of compounds in EBC. The aim of this study was to investigate methodological issues for measuring nitric oxide metabolites (NO(x)) in EBC. Twenty-five healthy adults (12 females, age range 23-55 years) and 22 children (11 females, age range 7-6 years) were recruited for studies investigating inter- and intra-day repeatability, repeatability with controlled expiratory flows and temperature, flow dependence, and analytical variability of EBC NO(x). Both intra- and inter-day repeatability was poor with a coefficient of repeatability of 103.4% of the mean difference between intra-day (15 min) measures and 118.6% of inter-day (24 h) differences. Repeatability was not improved when expiratory flow and temperature of the collection device were controlled. However, some of the variability (approximately 50%) may be accounted for by variability in the analytical technique (analytical variability) and this may result from difficulties in controlling for contamination. NO(x) levels were not affected by different expiratory flows in either adults or children but there was still significant variation within individuals. Levels of NO(x) in EBC seem to be highly variable and this needs to be considered if EBC NO(x) is to be used in clinical studies.