PurposeThis study aims to identify common and distinct hemodynamic and functional connectivity (FC) features for self-rated fatigue and depression symptoms in patients with clinically isolated syndrome (CIS) and relapsing–remitting multiple sclerosis (RR-MS).MethodsTwenty-four CIS, 29 RR-MS patients, and 39 healthy volunteers were examined using resting-state fMRI (rs-fMRI) to obtain whole-brain maps of (i) hemodynamic response patterns (through time shift analysis), (ii) FC (via intrinsic connectivity contrast maps), and (iii) coupling between hemodynamic response patterns and FC. Each regional map was correlated with fatigue scores, controlling for depression, and with depression scores, controlling for fatigue.ResultsIn CIS patients, the severity of fatigue was associated with accelerated hemodynamic response in the insula, hyperconnectivity of the superior frontal gyrus, and evidence of reduced hemodynamics–FC coupling in the left amygdala. In contrast, depression severity was associated with accelerated hemodynamic response in the right limbic temporal pole, hypoconnectivity of the anterior cingulate gyrus, and increased hemodynamics–FC coupling in the left amygdala. In RR-MS patients, fatigue was associated with accelerated hemodynamic response in the insula and medial superior frontal cortex, increased functional role of the left amygdala, and hypoconnectivity of the dorsal orbitofrontal cortex, while depression symptom severity was linked to delayed hemodynamic response in the medial superior frontal gyrus; hypoconnectivity of the insula, ventromedial thalamus, dorsolateral prefrontal cortex, and posterior cingulate; and decreased hemodynamics–FC coupling of the medial orbitofrontal cortex.ConclusionThere are distinct FC and hemodynamic responses, as well as different magnitude and topography of hemodynamic connectivity coupling, associated with fatigue and depression in early and later stages of MS.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00234-023-03174-1.