Global Lung Function Initiative (GLI) has recently published international reference values for diffusing capacity of the lungs for carbon monoxide (DLCO). Lower limit of normal (LLN), i.e. the 5th percentile, usually defines impaired DLCO We studied if the GLI LLN for DLCO differs from the LLN in a Swedish population of healthy, never-smoking individuals and how any such differences affect identification of subjects with respiratory burden.Spirometry, DLCO, chest high-resolution computed tomography and questionnaires were obtained from the first 15 040 participants, aged 50-64 years, of the Swedish CArdioPulmonary bioImage Study (SCAPIS). Both GLI reference values and lambda-mu-sigma method were used to define LLN in asymptomatic never-smokers without respiratory disease (n=4903 whereof 2329 women).Both SCAPIS median and LLN for DLCO were above the GLI median and LLN (p<0.05). The prevalence of DLCO GLI LLN was 5.7%.Subjects with DLCO >GLI LLN, but LLN GLI and >LLN SCAPIS) (n=13 600). No differences were found with regard to physician-diagnosed asthma.GLI LLN for DLCO is lower than the estimated LLN in Swedish healthy, never-smoking middle-aged adults. Individuals with DLCO above GLI LLN, but below SCAPIS LLN, had to a larger extent an increased respiratory burden. This suggests clinical implications of choosing adequate LLN for studied populations.