Background: Stress and poor mental health are significant issues in the workplace and are a major cause of absenteeism and reduced productivity. Understanding what might contribute towards employee stress is important for managing mental health in this setting. Physical activity has been shown to be beneficial to stress but less research has addressed the potential negative impact of sedentary behaviour such as sitting. Therefore, the aim of this study was to assess the relationship between device-measured occupational desk-based sitting patterns and stress (hair cortisol levels (HCL), as a marker of chronic stress and self-reported perceived stress (PS)). Methods: Employees were recruited from four workplaces located in Central Scotland with large numbers of desk-based occupations. Seventy-seven participants provided desk-based sitting pattern data (desk-based sitting time/day and desk-based sit-to-stand transitions/day), a hair sample and self-reported perceived stress. HCL were measured using enzyme-linked immunosorbent assay and PS using the Cohen Self-Perceived Stress Scale. Linear regression models were used to test associations between desk-based sitting time/day, desk-based sit-to-stand transitions/day, HCL and PS. Results: There were no associations between any of the desk-based sitting measures and either HCL or PS. Conclusions: Desk-based sitting patterns in the workplace may not be related to stress when using HCL as a biomarker of chronic stress or PS. The relationship between sitting patterns and stress therefore requires further investigation.