Isatis tinctoria is an ancient dye and medicinal plant with potent anti-inflammatory and antiallergic properties. Metabolic differences were investigated by NMR spectroscopy of accessions from different origins that were grown under identical conditions on experimental plots. For these accessions, metabolite profiles at different harvesting dates were analyzed, and single and repeatedly harvested plants were compared. Leaf samples were shock-frozen in liquid N2 immediately after being harvested, freeze-dried, and cryomilled prior to extraction. Extracts were prepared by pressurized liquid extraction with ethyl acetate and 70% aqueous methanol. NMR spectra were analyzed using a combination of different methods of multivariate data analysis such as principal component analysis (PCA), canonical analysis (CA), and k-nearest neighbor concept (k-NN). Accessions and harvesting dates were well separated in the PCA/CA/k-NN analysis in both extracts. Pairwise statistical total correlation spectroscopy (STOCSY) revealed unsaturated fatty acids, porphyrins, carbohydrates, indole derivatives, isoprenoids, phenylpropanoids, and minor aromatic compounds as the cause of these differences. In addition, the metabolite profile was affected by the repeated harvest regime, causing a decrease of 1,5-anhydroglucitol, sucrose, unsaturated fatty acids, porphyrins, isoprenoids, and a flavonoid.