PURPOSE: Nuclear magnetic resonance (NMR) spectroscopy has been used to quantify lipid wax, cholesterol ester terpenoid and glyceride composition, saturation, oxidation, and CH₂ and CH₃ moiety distribution. This tool was used to measure changes in human meibum composition with meibomian gland dysfunction (MGD). METHODS: (1)H-NMR spectra of meibum from 39 donors with meibomian gland dysfunction (Md) were compared to meibum from 33 normal donors (Mn). RESULTS: Principal component analysis (PCA) was applied to the CH₂/CH₃ regions of a set of training NMR spectra of human meibum. PCA discriminated between Mn and Md with an accuracy of 86%. There was a bias toward more accurately predicting normal samples (92%) compared with predicting MGD samples (78%). When the NMR spectra of Md were compared with those of Mn, three statistically significant decreases were observed in the relative amounts of CH₃ moieties at 1.26 ppm, the products of lipid oxidation above 7 ppm, and the =CH moieties at 5.2 ppm associated with terpenoids. CONCLUSIONS: Loss of the terpenoids could be deleterious to meibum since they exhibit a plethora of mostly positive biological functions and could account for the lower level of cholesterol esters observed in Md compared with Mn. All three changes could account for the higher degree of lipid order of Md compared with age-matched Mn. In addition to the power of NMR spectroscopy to detect differences in the composition of meibum, it is promising that NMR can be used as a diagnostic tool.