Importance: Intraoperative stressors may compound cognitive load, prompting performance decline and threatening patient safety. However, not all surgeons cope equally well with stress, and the disparity between performance stability and decline under high cognitive demand may be characterized by differences in activation within brain areas associated with attention and concentration such as the prefrontal cortex (PFC). Objective: To compare PFC activation between surgeons demonstrating stable performance under temporal stress with those exhibiting stress-related performance decline. Design, Setting, and Participants: Cohort study conducted from July 2015 to September 2016 at the Imperial College Healthcare National Health Service Trust, England. One hundred two surgical residents (postgraduate year 1 and greater) were invited to participate, of which 33 agreed to partake. Exposures: Participants performed a laparoscopic suturing task under 2 conditions: self-paced (SP; without time-per-knot restrictions), and time pressure (TP; 2-minute per knot time restriction). Main Outcomes and Measures: A composite deterioration score was computed based on between-condition differences in task performance metrics (task progression score [arbitrary units], error score [millimeters], leak volume [milliliters], and knot tensile strength [newtons]). Based on the composite score, quartiles were computed reflecting performance stability (quartile 1 [Q1]) and decline (quartile 4 [Q4]). Changes in PFC oxygenated hemoglobin concentration (HbO2) measured at 24 different locations using functional near-infrared spectroscopy were compared between Q1 and Q4. Secondary outcomes included subjective workload (Surgical Task Load Index) and heart rate. Results: Of the 33 participants, the median age was 33 years, the range was 29 to 56 years, and 27 were men (82%). The Q1 residents demonstrated task-induced increases in HbO2 across the bilateral ventrolateral PFC (VLPFC) and right dorsolateral PFC in the SP condition and in the VLPFC in the TP condition. In contrast, Q4 residents demonstrated decreases in HbO2 in both conditions. The magnitude of PFC activation (change in HbO2) was significantly greater in Q1 than Q4 across the bilateral VLPFC during both SP (mean [SD] left VLPFC: Q1, 0.44 [1.30] μM; Q4, -0.21 [2.05] μM; P < .001; right VLPFC: Q1, 0.46 [1.12] μM; Q4, -0.15 [2.14] μM; P < .001) and TP (mean [SD] left VLPFC: Q1, 0.44 [1.36] μM; Q4, -0.03 [1.83] μM; P = .001; right VLPFC: Q1, 0.49 [1.70] μM; Q4, -0.32 [2.00] μM; P < .001) conditions. There were no significant between-group differences in Surgical Task Load Index or heart rate in either condition. Conclusions and Relevance: Performance stability within TP is associated with sustained prefrontal activation indicative of preserved attention and concentration, whereas performance decline is associated with prefrontal deactivation that may represent task disengagement.