Dopaminergic, opiod and endocannabinoid neurotransmission are thought to play an important role in the neurobiology of acute exercise and, in particular, in mediating positive affective responses and reward processes. Recent evidence indicates that changes in fractional amplitude of low-frequency fluctuations (zfALFF) in resting-state functional MRI (rs-fMRI) may reflect changes in specific neurotransmitter systems as tested by means of spatial correlation analyses. Here, we tested for this relationship at different exercise intensities in twenty young healthy trained athletes performing low-intensity (LIIE), high-intensity (HIIE) interval exercises and a control condition on three separate days. Positive And Negative Affect Schedule (PANAS) scores and rs-fMRI were acquired before and after each of the three experimental conditions. Respective zfALFF changes were analysed using a repeated measures ANOVAs. We explored spatial cross-correlations between pre-to-post zfALFF changes in each condition with available neurotransmitter maps using the JuSpace toolbox, and performed additional analyses for the main systems of interest (dopaminergic, opiod, endocannabinoid), focusing on specific brain networks related to ‘reward’ and ‘emotion’. Elevated PANAS Positive Affect was observed after LIIE and HIIE but not in the control condition. HIIE compared to the control condition resulted in differential zfALFF decreases in precuneus, orbitofrontal cortex, thalamus, and cerebellum, whereas differential zfALFF increases were identified in hypothalamus, pituitary, and periaqueductal gray. The spatial alteration patterns in zfALFF were positively associated with dopaminergic and μ-opioidergic receptor distributions within the ‘reward’ network. These findings provide new insight into the neurobiology of exercise supporting the importance of reward-related neurotransmission during high-intensity physical activity.KeypointsPositive mood changes, indexed as elevated PANAS Positive Affect, were identified after high- and low-intensity exercise bouts, supporting previous accounts on mood-improving effects of physical activity.High-intensity exercise was found to be associated with distributed changes in fractional amplitude of low-frequency fluctuations, indicating enduring neural activity changes after anaerobic exercise bouts.Results of spatial cross-correlations with representative PET neurotransmitter distribution maps suggest involvement of endocannabinoid, dopaminergic, and opioidergic neurotransmission after high-intensity exercise.Utilizing spatial cross-correlations of changes in fractional amplitude of low-frequency fluctuations and representative PET neurotransmitter distribution maps, despite being an indirect metric, provides an innovative methodological framework for human exercise research, as it allows for non-invasive testing of acute exercise-related changes multiple neurotransmitter.