Habitual long term physical activity is known to have beneficial cognitive, structural, and neuro-protective brain effects, but to date there is limited knowledge on whether a single session of exercise can alter the brain's functional connectivity, as assessed by resting-state functional magnetic resonance imaging (rs-fMRI). The primary objective of this study was to characterize potential session effects in resting-state networks (RSNs). We examined the acute effects of exercise on the functional connectivity of young healthy adults (N = 15) by collecting rs-fMRI before and after 20 min of moderate intensity aerobic exercise and compared this with a no-exercise control group (N = 15). Data were analyzed using independent component analysis, denoising and dual regression procedures. Regions of interest-based group session effect statistics were calculated in RSNs of interest using voxel-wise permutation testing and Cohen's D effect size. Group analysis in the exercising group data set revealed a session effect in sub-regions of three sensorimotor related areas: the pre and/or postcentral gyri, secondary somatosensory area and thalamus, characterized by increased co-activation after exercise (corrected p < 0.05). Cohen's D analysis also showed a significant effect of session in these three RSNs (p< 0.05), corroborating the voxel-wise findings. Analyses of the no-exercise dataset produced no significant results, thereby providing support for the exercise findings and establishing the inherent test-retest reliability of the analysis pipeline on the RSNs of interest. This study establishes the feasibility of rs-fMRI to localize brain regions that are associated with acute exercise, as well as an analysis consideration to improve sensitivity to a session effect.