Background: Multiple sclerosis is characterized by white matter lesions, which are visualized with conventional T2-weighted magnetic resonance imaging (MRI). Little is known about local metabolic processes preceding the appearance and during the pathological development of new lesions. Objective: To identify metabolite changes preceding white matter (WM) lesions and pathological severity of lesions over time. Methods: A total of 59 relapsing-remitting multiple sclerosis (MS) patients were scanned four times, with 6-month intervals. Imaging included short-TE magnetic resonance spectroscopic imaging (MRSI) and diffusion tensor imaging (DTI). Results: A total of 16 new lesions appeared within the MRSI slab in 12 patients. Glutamate increased (+1.0 mM (+19%), p = 0.039) 12 and 6 months before new lesions appeared. In these areas, the increase in creatine and choline 6 months before until lesion appearance was negatively correlated with radial diffusivity (ρ = −0.73, p = 0.002 and ρ = −0.72, p = 0.002). Increase in creatine also correlated with the increase of axial diffusivity in the same period (ρ = −0.53, p = 0.034). When splitting the lesions into “mild” and “severe” based on radial diffusivity, only mild lesions showed an increase in creatine and choline during lesion formation ( p = 0.039 and p = 0.008, respectively). Conclusion: Increased glutamate heralded the appearance of new T2-visible WM lesions. In pathologically “mild” lesions, an increase in creatine and choline was found during lesion formation.