AbstractRecent studies combining diffusion tensor-derived metrics and machine learning have shown promising results in the discrimination of multiple system atrophy (MSA) and Parkinson’s disease (PD) patients. This approach has not been tested using more complex methodologies such as probabilistic tractography. The aim of this work is assessing whether the strength of structural connectivity between subcortical structures, measured as the number of streamlines (NOS) derived from tractography, can be used to classify MSA and PD patients at the single-patient level. The classification performance of subcortical FA and MD was also evaluated to compare the discriminant ability between diffusion tensor-derived metrics and NOS. Using diffusion-weighted images acquired in a 3 T MRI scanner and probabilistic tractography, we reconstructed the white matter tracts between 18 subcortical structures from a sample of 54 healthy controls, 31 MSA patients and 65 PD patients. NOS between subcortical structures were compared between groups and entered as features into a machine learning algorithm. Reduced NOS in MSA compared with controls and PD were found in connections between the putamen, pallidum, ventral diencephalon, thalamus, and cerebellum, in both right and left hemispheres. The classification procedure achieved an overall accuracy of 78%, with 71% of the MSA subjects and 86% of the PD patients correctly classified. NOS features outperformed the discrimination performance obtained with FA and MD. Our findings suggest that structural connectivity derived from tractography has the potential to correctly distinguish between MSA and PD patients. Furthermore, NOS measures obtained from tractography might be more useful than diffusion tensor-derived metrics for the detection of MSA.