ABSTRACT Introduction/Purpose Head impacts associated with American football have been associated with short- and long-term disrupted cognitive performance. In this study, we aimed to determine the effects of an acute game of American football on cognitive function, memory, morphological changes, and functional connectivity in the brain. Methods Twelve members (mean ± standard deviation: age, 26.8 ± 5.2 yr; height, 183 ± 6.5 cm; weight, 98.8 ± 23.6 kg) of the Israel National American Football team were assessed 1 wk before (PRE) and 24-h post (24P) game. No head injuries to any of the players participating in the study occurred. All study participants were scanned using a Siemens Prisma 3-T magnetic resonance imaging (MRI) scanner to determine changes in resting-state functional MRI (fMRI) by measuring each participant’s connectivity of subcortical regions. In addition, brain-derived neurotrophic factor (BDNF) concentration and cognitive function were measured at each assessment. Results A significant increase in BDNF concentration (33%, P = 0.007) was noted at 24P. In addition, significant increases (p < 0.05) in cerebral connectivity within the hippocampus, amygdala, and the connections between the hippocampus and the somatomotor areas were noted between PRE and 24P. Elevations in hippocampus connectivity were correlated with changes in acute memory (r = 0.72, P = 0.03), and decreases in somatomotor–hippocampus connectivity were significantly correlated with a greater change in BDNF concentration (r = −0.67, P = 0.01). No significant changes were found in brain volume, cognitive function, or between brain connectivity and other regions. Conclusions The fMRI results suggested that, even during an American football game in which no known head injury occurred, changes in neurotrophin concentrations were correlated with morphological changes in the parietal and frontal regions of the brain.