Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature
June 19th, 2019
The purpose of this paper is to identify changes in stress levels after visitation to three different settings and to describe the methods used in identifying stress levels based on measures of salivary cortisol, α-amylase, and a psychological self-report instrument.
Here we present a protocol, used to examine the effects on levels of biophysical and psychological stress following a visitation to three locations with different levels of nature. We're interested to learn whether a site with higher levels of naturalness will be more effective in helping participants to reduce their stress levels. In addition to using self report questionnaire, the main advantage of using this technique is that analyzing levels of cortisol and amylase collected from participants saliva samples can give us a more accurate read and better understanding of how our body actually responds after visitation to recreation sites.
Demonstrating the procedure will be Dr.Hocevar, a professor from Indiana University. Begin by introducing yourself to the subject. Provide them with a consent form to sign explaining the voluntary nature, purpose, and procedures of the study.
Give the subject a red armband for future identification. Obtain physiological and psychological measures of stress levels via saliva samples, just prior to the recreational experience, and immediately following conclusion of the experience. Collect a one to two milliliter saliva sample using a passive drool method.
Instruct subjects to allow saliva to pool in their mouths, then drool down the two inch straw and into the cryo vial until one milliliter has been collected. Label samples with an assigned three digit ID number and letter to indicate the timing of data collection. Then, temporarily freeze the sample in a steroid foam box full of dry ice for no more than two hours.
Transport the marked samples to a laboratory and store at minus 80 degrees Celsius until analyzed. Finally, use a liquid amylase reagent set to quantify alpha amylase in saliva samples. Then, use a multi mode reader capable of reading an optical density at 405 nanometers with the temperature controlled to 37 degrees Celsius during the assay.
First, acquire a cortisol enzyme amino acid kit to quantify cortisol in saliva samples, a spectrophotometer to read an optical density, or OD, at 450 nanometers as well as software capable of using OD recordings from the plate reader to perform four parameter logistic curve fitting. Allow all reagents to equilibrate to 20 to 25 degrees Celsius for a minimum of 30 minutes. Dilute the cortisol assay buffer one to five using deionized water, then dilute the wash buffer one to 20 using deionized water.
Next, thaw the samples on ice prior to analysis. Then, dilute samples one to 10 with cortisol assay buffer and use within two hours of preparation. To prepare the standards, label glass test tubes one through seven.
Then, pipet 225 microliters of assay buffer into tube number one, and 125 microliters of buffer into tubes two through seven. Next, add 25 microliters of the cortisol stock solution to tube number one, and vortex. Finally, remove 125 microliters of buffer from tube number one and add it to tube number two, then vortex again.
Results indicated a decrease in significant levels of salivary cortisol after visiting site A, the natural setting. No significant changes in levels of cortisol were observed in site B and C.Further, levels of alpha amylase increased after subjects visited site C.After visitation to the three locations and using the self report questionnaires, significant decreases on the factors of demands and worry were observed. No significant changes in the factor tension was observed for any of the three locations and significant increases were reported at sites A and site B for the factor joy.
Typically, much of the research done on stress reduction has relied on self report questionnaires. In this study, we collected participant's saliva samples to analyze how our body actually responds after visitation to three sites with different levels of naturalness. In this visualization, we featured both a laboratory techniques and one example of the in filled data collection.
This technique allows researcher to capture our body's reaction to different environments. In confirms our hypothesis that natural environment can have positive effects on human health. Future research is suggested to apply biophysical measurements of level of stress across different natural environments or different outdoor activities with true experimental design.
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Overview
This study investigates the impact of visiting three different natural settings on stress levels, utilizing both physiological and psychological measures. The focus is on understanding how varying levels of naturalness in environments can influence stress reduction.
Key Study Components
Area of Science
- Neuroscience
- Psychology
- Environmental Science
Background
- Stress can significantly affect mental and physical health.
- Natural environments are believed to have restorative effects on stress.
- Salivary cortisol and α-amylase are biomarkers for stress assessment.
- Self-report questionnaires provide subjective measures of stress levels.
Purpose of Study
- To identify changes in stress levels after visitation to different settings.
- To describe methods for assessing stress through physiological and psychological measures.
- To determine if higher naturalness in environments leads to greater stress reduction.
Methods Used
- Participants visit three locations with varying levels of naturalness.
- Saliva samples are collected to measure cortisol and α-amylase levels.
- Participants complete a psychological self-report questionnaire.
- Dr. Hocevar demonstrates the procedure and ensures informed consent.
Main Results
- Changes in cortisol and α-amylase levels are analyzed post-visitation.
- Self-reported stress levels are compared across different settings.
- Findings indicate a correlation between naturalness and stress reduction.
- The study highlights the effectiveness of combining physiological and psychological assessments.
Conclusions
- Natural environments can play a crucial role in stress management.
- Utilizing both biological and psychological measures provides a comprehensive understanding of stress responses.
- Further research is needed to explore long-term effects of nature exposure on stress.
Frequently Asked Questions
Executive Industry Relevance
This protocol demonstrates a dual-modality approach to stress assessment that combines biophysical biomarkers with psychological self-reports, offering a more comprehensive evaluation of environmental interventions. For biopharma R&D, such integrated measurement strategies support target validation in neuropsychiatric and psychosomatic research by providing objective, quantifiable endpoints that reduce reliance on subjective reporting alone. The method enables mechanistic de-risking of environmental or behavioral therapeutics by establishing reproducible, translatable stress-response profiles across controlled conditions.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying changes in cortisol and α-amylase as objective indicators of stress pathway modulation.
- Operational Value: Supports biological de-risking through standardized saliva collection and ELISA-based quantification, reducing variability in preclinical stress models.
- Predictive Value: Facilitates portfolio triage by linking environmental exposure to measurable biomarker shifts, improving confidence in target engagement for stress-related indications.
Screening & Assay Development
- Scientific Value: Prepares validated biological matrices (saliva) for downstream biomarker screening, ensuring assay readiness for cortisol and α-amylase detection.
- Operational Value: Addresses reproducibility through standardized drool collection, labeling, and cold-chain handling, enabling scalable sample processing across sites.
- Assay Readiness: Highlights compatibility with multi-mode plate readers and kinetic OD readings at 405 nm and 450 nm, supporting high-throughput biomarker quantification.
Translational & Preclinical Research
- Translational Continuity: Connects discovery-phase stress modulation observations to preclinical validation by using clinically relevant biomarkers (cortisol, α-amylase) with established roles in HPA axis and sympathetic nervous system activity.
- Risk-Adjusted Advancement: Supports go/no-go decisions by demonstrating significant biomarker changes in high-nature settings versus minimal change in low-nature sites, providing a gradient of biological response.
- Mechanistic De-risking: Focuses on predictive confidence by isolating the independent variable (environmental naturalness) and measuring dependent variable outputs (biomarker levels, PSQ factors) to clarify mechanism of action.
Pipeline & Workflow Integration
The method fits within the early discovery continuum, supporting hypothesis testing in environmental neuroscience and enabling progression to lead identification through quantifiable stress-response phenotypes.
- Discovery Biology: Supports pathway clarification by measuring cortisol (HPA axis) and α-amylase (sympathetic activation) as downstream readouts of stress modulation following environmental exposure.
- Screening: Describes assay readiness via standardized saliva collection, labeling, and storage at −80°C, ensuring reproducible biomarker quantification across experimental groups.
- Analytics: Highlights ELISA-based cortisol quantification and enzymatic α-amylase detection as quantitative outputs that enable inter-group comparison and statistical analysis of stress reduction.
- Translational Research: Connects biomarker changes to preclinical continuity by using salivary cortisol and α-amylase—analytes with established relevance in stress-related disease models.
- Enterprise Reuse: Positions the saliva collection and biomarker assay workflow as a reusable platform for evaluating diverse environmental, behavioral, or pharmacological interventions targeting stress pathways.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence through dual biomarker measurement, reduction of mechanistic ambiguity in stress response pathways.
- Operational Value: Standardization, reproducibility, and scalability of saliva-based biomarker collection and analysis across multiple field sites.
- Strategic Value: Better go/no-go decisions, capital efficiency, and reduced late-stage biological risk by identifying effective environmental modifiers early in discovery.
- Portfolio Impact: Risk-adjusted prioritization based on biomarker response gradients across naturalness levels, enabling data-driven advancement of promising interventions.
Implementation Considerations
- Required expertise in salivary biospecimen collection, biomarker assay execution (ELISA, enzymatic kinetics), and psychological questionnaire administration.
- Instrumentation needs include multi-mode plate readers capable of OD readings at 405 nm and 450 nm, temperature-controlled incubation at 37°C, and cold-chain storage (−80°C) for sample integrity.
- Cross-team standardization requires alignment between field collection staff, laboratory technicians, and data analysts on timing, labeling, and assay protocols.
- Adaptation considerations include validating saliva collection and biomarker stability across diverse outdoor conditions, subject populations, and environmental stressors.
- Practical limitations include the 45-minute post-collection processing window for saliva samples and the need for controlled timing relative to environmental exposure, as noted in the protocol.
