Recently introduced horizontal attenuated total reflectance (HATR) Fourier transform infrared (FTIR) spectroscopy for real-time assessment and continuous monitoring of glucose biomolecules in the skin tissue directly on the patients might appear a promising alternative to interpret the activity of interstitial glucose metabolism in vivo by means of evaluating the dynamics of changes of glucose concentrations in interstitial fluid (IF). In the present study, in vivo spectra by ATR-FTIR spectroscopy were obtained post-prandially during a 120–180-minute continuous monitoring in three patients with type 2 diabetes and compared to pre-prandial spectra. In all patients with diabetes interstitial glucose levels at 1030 and 1041cm^-1 reflected the best relationship with blood glucose. The lag time (LT) required for glucose to diffuse from the capillary to epidermal skin tissue was calculated between 0 and 60 minutes at all measured glucose biomolecules. Data showed intra- and inter-subject variations of each glucose biomolecule, pointing to similarities and differences among interstitial glucose metabolism of the patients. Finally, the findings suggest that HATR-FTIR spectroscopy might have the potential for clinical interpretation of activity of glucose metabolism for diagnosis, management, and treatment of patients with diabetes.