Neuroimaging investigations in non-exercise contexts have shown that the dorsolateral prefrontal cortex (dlPFC), medial PFC and anterior cingulate, are engaged when individuals attempt to cognitively control negative affect. Moreover, there are indications that aversive interoceptive stimuli preferentially activate the right hemisphere. We theorized that affective responses to incremental exercise would be regulated by the same prefrontal network implicated in non-exercise affect regulation. We hypothesized that there would be preferential right-dlPFC activation, among individuals with low tolerance to exercise intensity and, therefore, less positive affective responses to challenging intensities of exercise (i.e., above ventilatory threshold, VT). PURPOSE: To investigate dlPFC activation and affective responses during incremental exercise. METHODS: Thirty-eight participants (15M, 21F, Age: 23.7 ± 6.9 y; BMI: 24.0 ± 4.8 kg·m-2; VO2max: 32.8 ± 7.8 ml·kg-1·min-1) completed an incremental cycling test to volitional termination. They were divided into low- and high-Tolerance groups based on a median split of their Tolerance scores (Preference for and Tolerance of the Intensity of Exercise Questionnaire). Near-infrared spectroscopy was used to assess changes from rest in the Tissue Oxygenation Index (ΔTOI) in the left (AF3) and right (AF4) dlPFC. Affective valence ratings (Feeling Scale; FS) were collected each min. RESULTS: Tolerance scores were positively correlated with FS ratings above VT (r = 0.33, p =.04), such that lower-Tolerance individuals reported lower FS ratings. For ΔTOI, a significant interaction was found between Tolerance group (low-high) and Hemisphere (left-right), p =.02, ηp2 =.129. ΔTOI in the right dlPFC was larger for low- vs high-Tolerance individuals (p =.03). CONCLUSION: Low self-reported tolerance for exercise intensity is associated with lower ratings of affective valence above VT and larger increases in right-dlPFC oxygenation from rest. These results suggest that the prefrontal regulation of negative affective responses to increasing exercise intensity may exhibit similarities to the regulation of negative affective responses in non-exercise contexts.