Multiple sclerosis (MS) is a primary inflammatory demyelinating disease associated with a probably secondary progressive neurodegenerative component. Impaired mitochondrial functioning has been hypothesized to drive neurodegeneration and to cause increased anaerobic metabolism in MS. The aim of our multicentre study was to determine whether MS patients had values of circulating lactate different from those of controls. Patients (n = 613) were recruited, assessed for disability and clinically classified (relapsing–remitting, secondary progressive, primary progressive) at the Catholic University of Rome, Italy (n = 281), at the MS Centre Amsterdam, The Netherlands (n = 158) and at the S. Camillo Forlanini Hospital, Rome, Italy (n = 174). Serum lactate levels were quantified spectrophotometrically with the analyst being blinded to all clinical information. In patients with MS serum lactate was three times higher (3.04 ± 1.26 mmol/l) than that of healthy controls (1.09 ± 0.25 mmol/l, p < 0.0001) and increased across clinical groups, with higher levels in cases with a progressive than with a relapsing–remitting disease course. In addition, there was a linear correlation between serum lactate levels and the expanded disability scale (EDSS) (R2 = 0.419; p < 0.001). These data support the hypothesis that mitochondrial dysfunction is an important feature in MS and of particular relevance to the neurodegenerative phase of the disease. Measurement of serum lactate in MS might be a relative inexpensive test for longitudinal monitoring of “virtual hypoxia” in MS and also a secondary outcome for treatment trials aimed to improve mitochondrial function in patients with MS.