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Glycated keratin allows the monitoring of average tissue glucose exposure over previous weeks. In the present study, we wanted to explore if near-infrared (NIR) spectroscopy could be used as a non-invasive diagnostic tool for assessing glycation in diabetes mellitus.A total of 52 patients with diabetes mellitus and 107 healthy subjects were enrolled in this study. A limited number (n=21) of nails of healthy subjects were glycated in vitro with 0.278 mol/L, 0.556 mol/L and 0.833 mol/L glucose solution to study the effect of glucose on the nail spectrum. Consequently, the nail clippings of the patients were analyzed using a Thermo Fisher Antaris II Near-IR Analyzer Spectrometer and near infrared (NIR) chemical imaging. Spectral classification (patients with diabetes mellitus vs. healthy subjects) was performed using partial least square discriminant analysis (PLS-DA).In vitro glycation resulted in peak sharpening between 4300 and 4400 cm−1 and spectral variations at 5270 cm−1 and between 6600 and 7500 cm−1. Similar regions encountered spectral deviations during analysis of the patients’ nails. Optimization of the spectral collection parameters was necessary in order to distinguish a large dataset. Spectra had to be collected at 16 cm−1, 128 scans, region 4000-7500 cm−1. Using standard normal variate, Savitsky-Golay smoothing (7 points) and first derivative preprocessing allowed for the prediction of the test set with 100% correct assignments utilizing a PLS-DA model.Analysis of protein glycation in human fingernail clippings with NIR spectroscopy could be an alternative affordable technique for the diagnosis of diabetes mellitus.