AbstractA growing number of studies have revealed that higher aerobic fitness is associated with better working memory (WM) performance in older adults. However, the underlying functional neural mechanisms of this association remain under debate. It has been reported that aging increases recruitment of the prefrontal cortex (PFC) during cognitive tasks, and that this is associated with task performance in a compensatory manner. Therefore, this study aimed to clarify the prefrontal activation pattern that is associated with the relationship between aerobic fitness and WM performance in older adults, focusing on age-related extended prefrontal recruitment. Forty-seven older adults (65–74 years, 29 females) and 49 younger adults (18–24 years, 23 female) performed verbal and spatial n-back tasks, which included 0-, 1-, and 2-back conditions. Reaction time (RT) and accuracy (ACC) were assessed as indices of task performance. Prefrontal activation during the experimental tasks was monitored using functional near-infrared spectroscopy (fNIRS) and analyzed using an adaptive GLM method. We compared task performance and prefrontal activation between age groups to find age-related prefrontal activation patterns. Only older adults underwent a graded exercise test (GXT) to determine their ventilation thresholds (VT) as a measure of aerobic fitness, and, subsequently, the relationships among aerobic fitness, n-back task performance, and prefrontal activation in older adults were examined using correlation analysis and mediation analysis controlling for possible covariates. A comparison of task performance between groups revealed that older adults had slower RT and lower ACC than did younger adults, especially in the higher WM load 2-back condition. Group comparisons of prefrontal activation showed that older adults exhibited additional or greater activation than younger adults mainly in the ventrolateral PFC (VLPFC) and front polar area (FPA) in both the verbal and spatial 2-back conditions. Correlation analysis showed a relationship between higher VT, shorter RT for the verbal 2-back condition, and greater prefrontal activation of the bilateral FPA and right VLPFC during verbal 2-back conditions in older adults. In addition, mediation analyses indicated the possibility of a mediation effect of the prefrontal activation on the relationship between VT and RT for the verbal 2-back condition. These results suggest that older adults with higher aerobic fitness levels recruited more extended PFC regions, possibly for compensatory activation, to enhance their performance of the verbal n-back task. This study sheds light on the neural mechanisms underpinning the relationship between aerobic fitness and cognitive function in older adults.