The acute stress response is characterized by activation of multiple interconnected systems in the body, resulting in the release of a flood of hormones and immune mediators into circulation. In addition to detection of these molecules in the serum, saliva can serve as a source of these markers as well and can be collected in a non-invasive way. The complete profile of salivary biomarkers associated with the hypothalamic pituitary adrenal/gonadal axes and the immune system during the acute stress response has not been fully elucidated. In a cohort of 62 first responders engaged in a stress training exercise, we set out to determine patterns of cytokine, chemokine and hormone shifts during the acute stress response. Salivary samples were collected immediately before (pre-stress), immediately after (post-stress) and 1 h after the stress test (recovery). Multiplex ELISA panels of 42 cytokines and 6 steroid and thyroid hormones were used to determine concentrations of these biomarkers during the three aforementioned time points. Principal components analysis was conducted to determine patterns in the large data sets collected. In our ≥0.3 loading principal components analysis, for pre-stress vs. post, post-stress vs. recovery and pre-stress vs. recovery, a total of three, four and three factors accounted for 56.6, 68.34, and 61.70% of the biomarker variation for each phase respectively. In the ≥0.7 loading principal components analysis, three, four and three factors were found for pre-stress vs. post, post-stress vs. recovery and pre-stress vs. recovery stages, respectively. Of note, in our ≥0.3 loading principal components analysis, MCP1 was present in all three factors from pre-stress to post-stress, and fractalkine was found to be in all four factors post-stress vs. recovery and pre vs. recovery from stress. Additionally, hormones testosterone, estradiol, T4 and T3 grouped together consistently in the same factor for all phases of acute stress in both ≥0.3 and ≥0.7 principal components analysis. Overall, our results identified specific patterns of immune markers and hormones that shift during acute stress and warrant further investigation to understand their mechanistic role in regulating the stress response.