This pilot study is one of the first to examine the impact of job strain and shift work on both the autonomic nervous system (ANS) and the hypothalamic-pituitary-adrenal (HPA) axis using two salivary stress biomarkers and two subclinical heart disease indicators. This study also tested the feasibility of a rigorous biological sampling protocol in a busy workplace setting. Paramedics (n = 21) self-collected five salivary samples over 1 rest and 2 workdays. Samples were analyzed for α-amylase and cortisol diurnal slopes and daily production. Heart rate variability (HRV) was logged over 2 workdays with the Polar RS800 Heart Rate monitors. Endothelial functioning was measured using fingertip peripheral arterial tonometry. Job strain was ascertained using a paramedic-specific survey. The effects of job strain and shift work were examined by comparing paramedic types (dispatchers vs. ambulance attendants) and shift types (daytime vs. rotating day/night). Over 90% of all expected samples were collected and fell within expected normal ranges. Workday samples were significantly different from rest day samples. Dispatchers reported higher job strain than ambulance paramedics and exhibited reduced daily alpha-amylase production, elevated daily cortisol production, and reduced endothelial function. In comparison with daytime-only workers, rotating shift workers reported higher job strain, exhibited flatter α-amylase and cortisol diurnal slopes, reduced daily α-amylase production, elevated daily cortisol production, and reduced HRV and endothelial functioning. Despite non-statistically significant differences between group comparisons, the consistency of the overall trend in subjective and objective measures suggests that exposure to work stressors may lead to dysregulation in neuroendocrine activity and, over the long-term, to early signs of heart disease. Results suggest that further study is warranted in this population. Power calculations based on effect sizes in the shift type comparison suggest a study size of n = 250 may result in significant differences at p = 0.05. High compliance among paramedics to complete the intensive protocol suggests this study will be feasible in a larger population.