Becoming a first-time parent is meaningful and exciting, however the transition to parenthood (TTP) also tends to be associated with increases in stress and decreases in relationship satisfaction. Both stressful life events and relationship problems have been shown to have significant and negative impacts on physiological health, but research has yet to investigate the physiological stress response across the TTP, particularly in the context of the new parents’ relationship. The present study investigated whether new parents experience change in physiological stress, measured by diurnal cortisol slopes, across the TTP and whether relationship conflict and emotions differentially affected their stress response across this transition. Using a sample of 62 couples expecting their first child who completed 21-daily diaries assessing relationship variables at three time points across the TTP: pregnancy, newborn (2-3 weeks), and infancy (15 weeks). Couples also provided morning and evening saliva samples for 3 days in each stage to assess diurnal cortisol. I hypothesized that couples’ cortisol slopes would become less healthy across the TTP. I also predicted that those whose cortisol slopes became less healthy across the TTP would also experience a greater decline in relationship satisfaction. I further hypothesized that on days when couples reported more negativity in their relationship, they would exhibit less healthy diurnal cortisol slopes and that this association would become stronger across the transition to parenthood. Finally, I predicted that those who were most reactive to relationship negativity would show greater declines in relationship satisfaction. Results for the current study didn’t support my hypotheses: cortisol slopes actually became healthier for both new mothers and partners. Additionally, daily negative and positive relationship feelings, and daily conflict were not significantly associated with diurnal cortisol across the TTP. Nor was I able to detect variation in individuals’ cortisol slopes and was therefore unable to examine whether variation was predictive of relationship satisfaction. The current study highlights the difficulties with collecting cortisol in an uncontrolled (i.e. home) environment, as well as a lack of consistency in the literature regarding which variables are important to control/exclude when analyzing diurnal cortisol slopes.