<b><i>Background:</i></b> Characterizing long-term diffusing capacity (D<smlcap>l</smlcap>_CO) variability is important in assessing quality control for D<smlcap>l</smlcap>_CO equipment and patient management. Long-term D<smlcap>l</smlcap>_CO variability has not been reported. <b><i>Objectives:</i></b> It was the aim of this study to characterize long-term variability of D<smlcap>l</smlcap>_CO in a cohort of biocontrols and to compare different methods of selecting a target value. <b><i>Methods:</i></b> Longitudinal D<smlcap>l</smlcap>_CO monitoring of biocontrols was performed as part of the inhaled insulin development program; 288 biocontrols were tested twice monthly for up to 5 years using a standardized technique. Variability, expressed either as percent change or D<smlcap>l</smlcap>_CO units, was assessed using three different target values. <b><i>Results:</i></b> The 90th percentile for mean intersession change in D<smlcap>l</smlcap>_CO was between 10.9 and 15.8% (2.6–4.1 units) depending on the target value. Variability was lowest when the mean of all D<smlcap>l</smlcap>_CO tests was used as the target value and highest when the baseline D<smlcap>l</smlcap>_CO was used. The average of the first six D<smlcap>l</smlcap>_CO tests provided an accurate estimate of the mean D<smlcap>l</smlcap>_CO value. Using this target, the 90th percentile for mean intersession change was 12.3% and 3.0 units. Variability was stable over time and there were no meaningful associations between variability and demographic factors. <b><i>Conclusions:</i></b> D<smlcap>l</smlcap>_CO biocontrol deviations >12% or >3.0 units, from the average of the first six tests, indicate that the instrument is not within quality control limits and should be carefully evaluated before further patient testing.