In semelparous Pacific salmon, increased cortisol levels accompany sexual maturation and may be related to the rapid senescence and death that occur after spawning. In fish with extremely high cortisol, pre-spawning mortality is more likely. This may be because elevated cortisol is accompanied by energy depletion and reduces the immune capacity of sexually maturing individuals, thus increasing their susceptibility to parasites and pathogens. Several studies have measured cortisol levels in Pacific salmon during the last few weeks prior to spawning, but there is a lack of information regarding longer-term changes in resting and stressed cortisol levels of migrating adult Pacific salmon. A better understanding of the scope of the cortisol response during sexual maturation could contribute to understanding the extremely high pre-spawning mortality experienced by some threatened populations of Pacific salmon. The objective of this study was to determine the scope of the cortisol stress response in spring Chinook salmon as well as the dynamics of resting cortisol during maturation. We conducted a laboratory experiment in which plasma cortisol in adult Chinook salmon was measured before and after application of a standardized stressor applied at monthly intervals during the last three months prior to spawning. We found that resting and stressed cortisol levels increased during the last three months prior to spawning. We also found that sexually maturing Chinook salmon are able to mount a cortisol response to acute stressors when resting levels are elevated during maturation. Additionally, the effects of stress and time on cortisol dynamics differed between males and females as well as between individuals, which has implications for population resilience to anthropogenic stressors in wild populations.