Beta2-microglobulin (β2-MG) is a pharmacodynamic marker of interferon-β activity in multiple sclerosis (MS). Its role in the natural course of the disease is not fully known. We analyzed the spontaneous fluctuation of β2-MG in free-treatment MS patients during a short-time course to quantify β2-MG as a marker of disease activity/progression. Thirty MS patients were clinically assessed and imaged monthly over a 3-month period. Sera were collected concomitantly for the evaluation of β2-MG, by means of an enzyme-linked immunosorbent assay. Sera from 20 healthy individuals (HI) were drawn and used as controls. The Mann–Whitney test was used when appropriate and time effect on radiological and biological measures was assessed by means of the random effect models. Eight (26.7%) patients experienced a clinical relapse but three (10%) required steroid treatment. A reduction in the contrast-enhancing lesion load (P = 0.02) and a trend (P = 0.07) toward a decrease in brain parenchyma fraction were observed. Baseline levels of β2-MG were similar in patients and HI. Patients' β2-MG values increased over the 3-month time period (P = 0.05) but did not exceed those detected in HI at any time point. These results failed to demonstrate the validity of β2-MG as a surrogate marker of disease in MS.