BACKGROUND Few biomarkers of progressive multiple sclerosis (MS) are sensitive to change within the two-year time frame of a clinical trial. OBJECTIVE To identify biomarkers of MS disease progression with magnetic resonance spectroscopy (MRS) in secondary progressive MS (SPMS). METHODS Forty-seven SPMS subjects were scanned at baseline and annually for two years. Concentrations of N-acetylaspartate, total creatine, total choline, myo-inositol, glutamate, glutamine, and the sum glutamate+glutamine were measured in a single white matter voxel. RESULTS Glutamate and glutamine were the only metabolites to show an effect with time: with annual declines of (95% confidence interval): glutamate -4.2% (-6.2% to -2.2%, p < 10(-4)), glutamine -7.3% (-11.8% to -2.9%, p = 0.003), and glutamate+glutamine -5.2% (-7.6% to -2.8%, p < 10(-4)). Metabolite rates of change were more apparent than changes in clinical scores or brain atrophy measures. CONCLUSIONS The high rates of change of both glutamate and glutamine over two years suggest they are promising new biomarkers of MS disease progression.