To assess the applicability of magnetic resonance spectroscopy (MRS) for long-term follow-up of neurological diseases a longitudinal 1H-MRS study at 3 T was carried out on ten patients having relapsing-remitting multiple sclerosis (MS) who, after baseline examination, received interferon-beta (IFN) 1b. At 8-20 examinations within up to 34 months absolute concentrations of N-acetylaspartate (NAA), total creatine (tG), and choline-containing compounds (tCho) were determined in a large non-enhancing lesion and contralateral normal appearing white matter (NAWM). MR spectra were analyzed using a novel time domain-frequency domain method including non-parametric background characterization. For comparison at baseline, ten healthy controls were examined. The concentrations of tCho and tCr were found to be higher in MS brain than in control brain. Besides a non-significantly lower NAA concentration in lesions there were no concentration differences between lesions and NAWM. Over the follow-up period the measured metabolite concentrations exhibited a high variability. Most concentrations remained within this scatter, and statistical tests revealed significant fluctuations in the levels of metabolites in one case only. This stability of the metabolite concentrations over time might result from IFN therapy as for the spontaneous course of relapsing-remitting MS decreasing metabolite (NAA/tCr) ratios have been reported. The results further suggest that future treatment trials intending to use metabolite concentrations as a secondary outcome indicator use even longer observation periods and, besides group analysis of large cohorts, investigate the time behavior of selected single cases. The biochemical abnormalities found in NAWM emphasize the importance of analyzing both lesion and NAWM.