To investigate brain functional connectivity (FC) alterations in patients with clinically isolated syndromes (CIS) presenting without conventional brain MRI lesions, and to identify the FC differences between the CIS patients who converted to multiple sclerosis (MS) and those not converted during a 5-year follow-up. We recruited 20 CIS patients without conventional brain lesions, 28 patients with MS and 28 healthy controls (HC). Normalized voxel-based functional connectivity strength (nFCS) was determined using resting-state fMRI (R-fMRI) and compared among groups. Furthermore, 5-years clinical follow-up of the CIS patients was performed to examine the differences in nFCS between converters and non-converters. Compared to HC, CIS patients showed significantly decreased nFCS in the visual areas and increased nFCS in several brain regions predominately in the temporal lobes. MS patients revealed more widespread higher nFCS especially in deep grey matter (DGM), compared to CIS and HC. In the four CIS patients converting to MS, significantly higher nFCS was found in right anterior cingulate gyrus (ACC) and fusiform gyrus (FG), compared to non-converted patients. We demonstrated both functional impairment and compensation in CIS by R-fMRI. nFCS alteration in ACC and FG seems to occur in CIS patients at risk of developing MS. aEuro cent Both functional impairment and compensation occur in CIS without conventional brain lesions. aEuro cent MS patients revealed more widespread higher nFCS especially in deep grey matter. aEuro cent nFCS alteration may help stratifying CIS at risk of developing MS.