Cortisol is a biomarker for stress monitoring; however, the biomedical and clinical relevance is still controversial due to the complexity of cortisol secretion mechanisms and their circadian cycles as well as environmental factors that affect physiological cortisol level, which include individual mood and dietary intake. To further investigate this multifaceted relationship, a human pilot study examined cortisol concentration in sweat and saliva samples collected from 48 college-aged participants during aerobic exercise sessions along with mental distress and nutrition surveys. Enzyme-linked immunosorbent assays determined highly significant differences between apocrine-dominant sweat (AP), saliva before exercise (SBE), and saliva after exercise (SAE) cortisol concentration (AP-SBE: p = 0.0017, AP-SAE: p = 0.0102). A significantly greater AP cortisol concentration was detected in males compared to females (p = 0.0559), and significant SAE cortisol concentration differences were also recorded between recreational athletes and non-athletes (p = 0.044). However, Kessler 10 Psychological Distress Scale (K10) scores, an examination administered to deduce overall wellness, provided no significant differences between males and females or athletes and non-athletes in distress levels, which statistically signifies a direct relationship to cortisol was not present. For further analysis, dietary intake from all participants was considered to investigate whether a multiplexed association was prevalent between nutrition, mood, and cortisol release. Significant positive correlations between AP cortisol, SAE cortisol, K10 scores, and fat intake among female participants and athletes were discovered. The various machine learning algorithms utilized the extensive connections between dietary intake, overall well-being, sex factors, athletic activity, and cortisol concentrations in various biofluids to predict K10 scores. Indeed, the understanding of physiochemical stress response and the associations between studied factors can advance algorithm developments for cortisol biosensing systems to mitigate stress-based illnesses and improve an individual’s quality of life.

Colorimetric analysis, which relies on a chemical reaction to facilitate a change in visible color, is a great strategy for detecting cortisol, which is necessary to diagnose and manage the wide variety of diseases related to the hormone, because it is simple in design, inexpensive, and reliable as a standard cortisol analysis technique. In this study, four different colorimetric cortisol analyses that use various chromogens, which include sulfuric acid, Porter–Silber reagent, Prussian blue, and blue tetrazolium, are studied. Modifications to the classic Porter–Silber method are made by increasing the carbon content of the alcohol and adding gold nanoparticles, which result in a twofold increase in reaction rate and a slight decrease in the limit of detection (LoD). After a comparison of the reaction rate, LoD, dynamic range, characteristic peaks, and color stability of all methods, blue tetrazolium demonstrates a low LoD (97 ng/mL), broad dynamic range (0.05–2 μg/mL), and quick reaction rate (color development as fast as 10 min), which are well within the requirements for human biofluids. Cortisol in artificial saliva and sweat and in human sweat was determined while confirming that no excipients or other biomarkers interfered with the reactions. Twenty-one human sweat samples were tested using blue tetrazolium and revealed a significant difference between male and female apocrine cortisol concentrations and showed a highly significant difference between apocrine and eccrine cortisol concentrations. Colorimetric methods of cortisol can compete with existing electrochemical sensors because of their similar accuracy and detection range in certain wearable biosensor applications. The simplicity of colorimetric methods advances potential applications in skin-interfaced bio-electronics and point-of-care devices.