This study was funded in part through the Faculty Research Grant Program (FRGP) sponsored through the School of Public Health at Indiana University, Bloomington, IN. The authors would like to thank Dr. Alison Voight and Melissa Page for their editorial assistance and constructive comments.

This study follows the policies and guidelines of Human Research Protection Program of Indiana University Institutional Review Board.
1. Location selection
<ol>
	<li>Select the number of sites (n) based on different levels of nature. 
	NOTE: We chose three sites for our work. Using a continuum based on levels of &#34;naturalness&#34;, Site A was considered the most natural and is comprised of approximately 1,200 acres of wooded ridges bordering a lake and set within a deciduous forest. The most common activities include walking and wildlife watching. Site B was a 33-acre municipal park featuring walking paths, places for gatherings, playgrounds, and open field space for causal recreational activities. Site C was an urban, indoor exercise facility with the lowest level of naturalness. All three sites are in relatively close proximity to a medium-size city (estimated population of 46,000 people) in the midwestern United States.</li>
</ol>
2. Participant screening and preparation
<ol>
	<li>Complete participant recruitment before subjects engage in recreational experiences. 
	NOTE: For Sites A and B, subjects were approached by the researcher in the parking lot of the park entrance. For Site C, subjects were approached at the front desk of the indoor exercise facility. To control for differences in activity type, subjects recruited for both Sites A and B were predominately hiking, while for Site C the primary activity was running or walking on the indoor track.</li>
	<li>During recruitment, remind subjects to comply with the guidelines related to collecting salivary cortisol samples. They must not eat or drink 10 min before providing saliva samples. 
	NOTE: It should be noted that in this case, the saliva samples were not normalized for volume or dilution (concentration) -&#160;i.e. whether the participants were adequately hydrated and/or did hydration change between the sample collections.</li>
	<li>Give red armbands (or equivalently noticeable clothing) to each participant to allow for easy identification when emerging at the end of their engagements.</li>
	<li>Assign a 30-40 min time allotment so that all subjects across the three sites spend a similar amount of time in each site. Exclude subjects who spend substantially more or less time than the rest of the sample.</li>
	<li>Keep the type of activity engaged consistent. 
	NOTE: In this case, the &#34;medium&#34;&#160;activity was walking or hiking. Subjects who engaged in activities substantially different from walking or hiking were excluded from the sample. For example, subjects who were fishing, picnicking, or weight lifting were not included in the respective samples.</li>
</ol>
3. Conditions and experimental design
<ol>
	<li>Use a quasi-experimental pre-test/post-test design.</li>
	<li>After identifying subjects and upon their agreeing to participate, ask each subject to read and sign an IRB form explaining the voluntary nature, purpose, and procedures of the study.</li>
	<li>Following this process, give subjects a piece of clothing (e.g., red armband) for future identification and obtain physiological and psychological measures of stress levels (i.e., PSQ, 1-2 mL of saliva that is spit or drooled into a test tube). Collect samples from the late afternoon to early evening. 
	NOTE: These data were collected by researchers both 1) just before subjects entered the site and 2) immediately upon ending visitation to the site.</li>
</ol>
4. Saliva samples
<ol>
	<li>To avoid sample dilution, ask subjects not to eat, drink, or rinse mouths 10 min before providing saliva samples.</li>
	<li>Ask subjects to provide 1-2 mL saliva samples (excluding foam) just prior to the recreational experience and immediately following conclusion of the experience.</li>
	<li>Collect saliva samples using a passive drool method:
	<ol>
		<li>Provide subjects with a 2 in plastic drinking straw to drool into a 2 mL cryovial (see Table of Materials).</li>
		<li>Instruct subjects to allow saliva to pool in their mouths, then drool down the straw and into the cryovial. According to the Saliva Collection and Handling Advice (2011), 1 mL (excluding foam) is adequate for most tests.</li>
		<li>Label samples with an assigned 3-digit ID number (i.e., 001) and letter to indicate the timing of data collection (i.e., A represents pre-test, B represents post-test; where 001A represents the saliva sample provided by participant 001 before conducting the recreational experience).</li>
		<li>After the sample is collected and labeled, it should then be stored frozen temporarily in a steroid foam box full of dry ice for no more than 2 h.</li>
		<li>Transport the marked samples to a laboratory and store at -80 &#176;C until analyzed.</li>
	</ol>
	</li>
</ol>
5. Quantification of &#945;-amylase
NOTE: In this assay, &#945;-amylase hydrolyzes 2-chloro-p-nitrophenyl-&#945;-D-maltotrioside to 2-chloro-nitrophenol and forms glucose, 2-chloro-p-nitrophenyl-&#945;-D-maltoside, maltotriose, and glucose. The reaction is monitored at an absorbance of 405 nm, which corresponds to &#945;-amylase activity in the sample. This assay demonstrates linearity between 0 and 2000 U/L.
<ol>
	<li>Materials
	<ol>
		<li>Used a liquid amylase reagent set (see Table of Materials) to quantify &#945;-amylase in saliva samples. All reagents are provided as ready-to-use liquids and stored at 0-4 &#176;C.</li>
		<li>Use a multi-mode reader (see Table of Materials) capable of reading an optical density at 405 nm with a temperature controlled to 37 &#176;C during the assay.</li>
	</ol>
	</li>
	<li>Analysis
	<ol>
		<li>Thaw the samples on ice prior to analysis.</li>
		<li>Dilute samples 1:10 with 1x PBS (10 &#181;L of saliva + 90 &#181;L of PBS).</li>
		<li>Analyze each sample in duplicate.</li>
		<li>Equilibrate the amylase reagent to 20-25 &#176;C for at least 30 min.</li>
		<li>Add 0.1 mL of amylase reagent to a 96 well microplate for each sample.</li>
		<li>Pre-incubate the microplate at 37 &#176;C for a minimum of 5 min.</li>
		<li>Add 2.5 &#181;L of the sample to the amylase reagent.</li>
		<li>Take an initial reading after 60 s.</li>
		<li>Continue readings every 60 s for an additional 2 min.</li>
		<li>Calculate the mean absorbance difference per minute (&#916;Abs/min).</li>
	</ol>
	</li>
	<li>Calculation
	<ol>
		<li>To calculate amylase activity, use the following formula: 
		<img alt="Equation 1" src="/files/ftp_upload/59272/59272eq1.jpg" /> 
		Where &#916;Abs/min = changes in absorbance difference per minute; TV = total assay volume (0.1025 mL); *1000 = conversion of U/mL to U/L; MMA = millimolar absorptivity of 2-chloro-p-nitrophenol = 12.9; SV = sample volume (0.0025 mL); and LP = light path (1 cm). Substituting gives: 
		<img alt="Equation 2" src="/files/ftp_upload/59272/59272eq2.jpg" /> 
		Therefore, multiply the &#916;Abs/min by 3178x the dilution factor (10) to obtain amylase in U/L.</li>
		<li>For samples above 2000 U/L (assay linearity) dilute further (at least 2x using PBS) and re-assay, then multiply the &#945;-amylase result by the additional dilution factor.</li>
	</ol>
	</li>
</ol>
6. Quantification of cortisol
NOTE: In this assay, free cortisol is quantified in saliva using a cortisol standard curve. Standards and diluted samples are added to a microtiter plate that is pre-coated with an antibody. A cortisol-peroxidase conjugate is added to the wells, followed by addition of a monoclonal antibody to cortisol. The amount of cortisol/peroxidase conjugate binding decreases as the concentration of cortisol in the sample increases.
<ol>
	<li>Materials
	<ol>
		<li>Use a Cortisol Enzyme Immunoassay Kit (see Table of Materials) to quantify cortisol in saliva samples. All reagents needed to perform this assay are included in the kit. All components of the kit are stored at 0-4 &#176;C prior to reaching the expiration date.</li>
		<li>Use a spectrophotometer able to read an optical density (OD) at 450 nm (see Table of Materials), as well as software capable of using OD recordings from the plate reader to perform four-parameter logistic curve (4 PLC) fitting.</li>
	</ol>
	</li>
	<li>Reagent preparation
	<ol>
		<li>Allow all reagents to equilibrate to 20-25 &#176;C for a minimum of 30 min.</li>
		<li>Dilute the cortisol assay buffer 1:5 using deionized water.</li>
		<li>Dilute the wash buffer 1:20 using deionized water. 
		NOTE: Assay and wash buffers are stable for 3 months when stored at 0-4 &#176;C.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Thaw the samples on ice prior to analysis.</li>
		<li>Dilute samples 1:10 with cortisol assay buffer (20 &#181;L of saliva + 180 &#181;L of buffer) and use within 2 h of preparation.</li>
	</ol>
	</li>
	<li>Preparation of standards
	<ol>
		<li>Label glass test tubes #1-#7.</li>
		<li>Pipet 225 &#956;L of assay buffer into tube #1 and 125 &#956;L of buffer into tubes #2-#7.</li>
		<li>Add 25 &#956;L of the cortisol stock solution to tube #1 and vortex.</li>
		<li>Remove 125 &#956;L of buffer from tube #1 and add it to tube #2, then vortex.</li>
		<li>Repeat the serial dilutions for tubes #3-#7. 
		NOTE: The final cortisol concentrations of each standard is shown in Table 1.</li>
		<li>Standards must be used within 2 h of preparation.</li>
	</ol>
	</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Standard</td>
			<td>#1</td>
			<td>#2</td>
			<td>#3</td>
			<td>#4</td>
			<td>#5</td>
			<td>#6</td>
			<td>#7</td>
		</tr>
		<tr>
			<td>Assay Buffer Volume (&#956;L)</td>
			<td>225</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
		</tr>
		<tr>
			<td>Addition</td>
			<td>Stock Std</td>
			<td>#1</td>
			<td>#2</td>
			<td>#3</td>
			<td>#4</td>
			<td>#5</td>
			<td>#6</td>
		</tr>
		<tr>
			<td>Volume of Addition (&#181;L)</td>
			<td>25</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
			<td>125</td>
		</tr>
		<tr>
			<td>Final Concentration (pg/mL)</td>
			<td>3200</td>
			<td>1600</td>
			<td>800</td>
			<td>400</td>
			<td>200</td>
			<td>100</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 1: Standard curve preparation table.
<ol start="5">
	<li>Analysis
	<ol>
		<li>Use the plate layout below in Figure 1 as a guide to set up the microplate.</li>
		<li>It is recommended to use a multichannel pipette for addition of reagents.</li>
		<li>Add 50 &#956;L of samples or standards into the appropriate number of wells in the plate. Samples and standards should be run in duplicate.</li>
		<li>Add assay buffer (75 &#956;L) into the non-specific binding (NSB) wells.</li>
		<li>Add assay buffer (50 &#956;L) into the maximum binding (B0) wells and zero standard (blank) wells.</li>
		<li>Add 25 &#956;L of the cortisol conjugate to each well.</li>
		<li>Add 25 &#956;L of the cortisol antibody to each well, except for the NSB wells.</li>
		<li>Gently tap the side of the plate to mix the reagents.</li>
		<li>Cover with the plate sealer and shake at room temperature (RT) at 20-25 &#176;C for 1 h.</li>
		<li>Remove the well contents and rinse each well 4x with wash buffer (300 &#924;l). Tap the plate to dry on absorbent towels between washes.</li>
		<li>Add the TMB substrate to each well (100 &#956;L).</li>
		<li>Incubate the plate at 20-25 &#176;C for 30 min without shaking.</li>
		<li>Add stop solution to each well (50 &#956;L).</li>
		<li>Read the optical density in each well of the microplate at 450 nm.</li>
		<li>Average the optical densities for each standard, then sample and subtract the mean optical density for the NSB wells.</li>
		<li>Calculate the % bound (B/B0) for all samples using maximum binding (B0) controls.</li>
		<li>Create a standard curve using software capable of four-parameter logistic regression curve fitting, calculated from the %B/B0 curve.</li>
		<li>Multiply the result by the dilution factor (10) to obtain cortisol values in pg/mL.</li>
		<li>Samples with optical densities falling above the highest standard should be further diluted with assay buffer and re-assayed, then the result should be multiplied by the additional dilution factor.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59272/59272fig1.jpg" /> 
Figure 1: Example plate layout. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
7. Psychological measurement (Perceived Stress Questionnaire)
<ol>
	<li>Measure psychological levels of subjects using the PSQ published by Fliege et al.13, which includes four factors (worry, tension, tension, joy) and utilizes 20 items.</li>
	<li>Ask the subject to fill out the PSQ just prior to their recreational experience and immediately following conclusion of the experience.</li>
	<li>Tag questionnaires with a 3-digit ID number identical to each subject&#39;s biophysiological level of stress.</li>
</ol>

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The authors declare no conflicts of interest.

The aim of this study is to identify potential changes in stress using biophysical and psychological instruments following recreational visitation to three different settings with different levels of nature. Both cortisol and &#945;-amylase have been shown to be reliable indicators of levels of psychological stress. The amylase assay procedure described in this study has been adapted to a 96 well format. When amylase levels in saliva are high, absorbance changes occur rapidly. Therefore, it is critical to limit the number of samples analyzed at one time, as the number of samples that can be analyzed at one time is limited by how quickly 2.5 &#181;L of the sample can be added to each well. In this study, amylase levels were measured in one column (eight reactions) at a given time. Two principal systems are involved in the stress response, including the hypothalamus-pituitary-adrenocortical axis (HPA) and the sympatho-adrenomedullary system (SAM). Kirschbaum and Hellhammer14 have reported that salivary cortisol measurements are closely correlated with serum cortisol levels and involve less hyper-stress complications connected with blood sampling techniques. Alpha-amylase is a major salivary enzyme typically associated with sympathetic (SAM) stimuli15 and considered to be a useful measurement tool for evaluating the SAM system16.
While both the biophysical and psychological measures are efficacious in detecting changes in levels of stress, several issues are present in this study. First, there was incongruity between the cortisol and &#945;-amylase measurement outcomes, with differences noted at different sites. Nater et al.17 reported that there is little evidence as to whether the HPA (cortisol) or SAM (amylase) is predominant during psychological stress. One potential explanation was made by Takai et al.18, who suggested that the process by which &#945;-amylase and cortisol enter the bloodstream with cortisol, indicating that a more complex and lengthy system is in operation.
Another explanation of the results involves the level and saliency of stress17. The results suggest that acute levels of stress result in greater association between cortisol and &#945;-amylase, and more moderate levels of stress result in greater disassociation. In this study, the stress experienced during recreational engagement are considered moderate at best. Thus, in studies such as the one presented here, in which experienced stress is low to moderate, differences in measured levels of cortisol and &#945;-amylase should be expected.
A third variable impacting the incongruity between the cortisol and &#945;-amylase measurements involves issues with salivary flow rate. There is limited evidence on the relationship between salivary flow rate, collection techniques, and stress19. In this study, saliva is collected via&#160; &#34;drooling&#34;&#160;into a test tube. Subjects are advised not to chew gum or eat before data collection, but how diligent they were in this study to these guidelines is unknown. Furthermore, using a salivette device may be more effective in collecting the required amount of saliva within a specified time. Nater et al.20 have suggested that there may be no differences in various biochemical characteristics based on whether cups or salivettes are used.
Finally, this study uses the &#34;drool&#34;&#160;method of collecting saliva as described by Granger et al.21. This method of collecting saliva has several advantages over other approaches but requires a competent, compliant, awake, and capable participant. As such, children under the age of six or elderly subjects are typically not considered suitable respondents. The advantages of this method include a large sample volume that facilitates assays for multiple markers and the fact that an unused sample can be frozen for future assays. In addition, the drool method minimizes effects from substances used to stimulate saliva flow such as chewing gum and drink mixes.
Another method for collecting data involves the use of cotton pledgets, where the saliva is absorbed by the pledget and expressed out of the cotton into a collection vial via centrigugation. Shirtcliff et al.19 issued a cautionary note stating that in certain situations, filtering saliva through cotton can cause interference in immunoassays. Other approaches include use of filter paper and hydrocellulose microsponges21. While each approach has specific advantages and disadvantages, given the sample size utilized in this study, the drool sampling method was chosen.
In conclusion, an accumulating body of research from a broad spectrum of disciplines suggest that natural environments can have positive effects on human health6,22. Typical of these types of settings include parks, greenspaces, gardens, and forested areas. Factors associated with these types of areas that are considered beneficial to health23 include improved air quality, heightened opportunities for physical activity and social contact, and enhanced feelings of quality of life. For example, Gidlow et al.24 found that while physical exercise had salutogenic effects in both natural and urban environments, natural settings were often more effective in reducing levels of stress.
Using a multiple-method approach involving biophysical measurements of cortisol and &#945;-amalyze and a self-report measuring perceived levels of stress, this study provides added support for the expanding body of literature suggesting that natural settings have beneficial effects on health-related issues such as reducing levels of stress25,26. This study also suggests that greater levels of nature have more pronounced potential benefits.
Several limitations exist in this study. The first is the fidelity of the biophysical data collection. While visitors were engaged in the saliva collection during similar times, namely mid- to late-afternoon through early evening, to account for the diurnal cycle of cortisol, researchers attempted to identify only those who had not eaten anything less than 2 h prior to collecting saliva. This was done via verbal questioning of when they had last ingested food. Thus, the researchers were dependent on the truthfulness of subject responses.
Second, because of the timing of data collection, response biases may have occurred within these samples, which may have been different if collection was done at a different time or by random selection. That is, subjects who visited each location in the afternoon or evening may not be representative of potential respondents who visited at different times.
Finally, while the biophysical data were collected and processed through well-recognized procedures, there was no measurement ascertaining chronic levels of stress. In this case, future studies should include a cortisol measurement using hair samples or similar techniques that determine long-term levels of stress before engagement in recreational activities at various locations.

Elevated stress levels have long been linked to many serious health conditions such as heart disease, obesity, and psychological disorders1,2,3. A growing body of research suggests that close proximity or visitation to natural settings such as park and non-developed landscapes can have a remarkable effect on psychological well-being and decreased levels of stress1,4,5,6,7,8,9,10. Explanations for the effects of natural settings and stress levels have included the following: (1) natural settings provide venues for physical activity8,11 and (2) visitors to natural environments have the ability to focus on more non-task thought processes, thereby leading to a reduction in attention fatigue12. To determine the effects of nature on stress reduction, this study utilizes a self-report of psychological stress (PSQ) and two saliva-based biomarkers, cortisol and &#945;-amylase, after visitation to three different recreation sites. These locations vary among their levels of &#34;naturalness&#34;&#160;and include a wilderness-type setting, municipal park, and local fitness and recreation facility.
This study aims to address the following research questions: (RQ1) Are there differences in levels of biophysical stress measured by salivary cortisol and &#945;-amylase when compared across all three sites (i.e., natural, semi-natural, built)? (RQ2) Are there differences in levels of psychological stress measured by the PSQ (manifested by four constructs: demands, worries, tension, and joy) when compared across all three sites (i.e., natural, semi-natural, built)?

<table><tbody><tr><td>Cortisol Enzyme Immunoassay Kit</td><td>DetectX</td><td>K003-H1</td><td>The Cortisol Enzyme Immunoassay kit is designed to quantitatively measure cortisol present in dried fecal extracts, saliva, urine, serum, plasma and culture media samples.</td></tr><tr><td>Cryogenic Labels for Cryogenic Storage</td><td>Fisherbrand</td><td>5-910-A</td><td>Unique adhesive withstands extreme temperature</td></tr><tr><td>Liquid Amylase (CNPG3) Reagent Set</td><td>Pointe Scientific</td><td>A7564</td><td>For the quantitative kinetic determination of &amp;alpha;-amylase activity in human serum.</td></tr><tr><td>Round Bottom 2mL Polypropylene Tubes with External Thread Cap</td><td>Greiner Bio-One</td><td>07-000-257</td><td>2.0 ml U-BTM Cryo.s self standing polypropylene sterilized</td></tr><tr><td>Synergy Multi-Mode Microplate Reader</td><td>BioTek</td><td></td><td>It is a single-channel absorbance, fluorescence, and luminescence microplate reader that uses a dual-optics design to perform measurements of samples in a microplate format.</td></tr></tbody></table>

measuring biophysical and psychological stress levels following visitation to three locations with differing levels of nature

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report formats, a growing number of studies now incorporate biological stress-related hormones and catalysts, such as cortisol and &#945;-amylase, to measure levels of stress. Presented here is a protocol to examine the effects on levels of biophysical and psychological stress following visitation to three different locations with differing levels of nature. Biophysical and self-reported psychological stress levels are measured immediately upon entering the selected locations and just prior to the visitors leaving the site. Using a &#34;drool&#34;&#160;method, the biophysical measure consists of 1-2 mL samples of saliva provided by study subjects upon entry to one of three study locations. As prescribed by extant literature, the saliva is collected within a 45 minute time frame following the end of the visitor&#8217;s engagement at the location. Following saliva collection, the samples are labeled and transported to a biological lab. Cortisol is the biophysical variable of interest in this study and measured using an ELISA process with a TECAN plate reader. To measure self-reported stress, the Perceived Stress Questionnaire (PSQ), which reports levels of worry, tension, joy, and perceived demands. Data are collected at all three sites in the late afternoon through early evening. When compared across all three settings, stress levels, as measured by both the biological markers and self-reports, are significantly lower after visitation to the most natural setting.

Sample Description 
Utilizing a quota sampling technique, this study recruited 35 visitors from each of the three sites. In total, 105 subjects were recruited in this study, including 63 males and 42 females. Average ages of visitors recruited from three different sites were 25.9 years (Site A), 37.2 years (Site B), and 28.8 years (Site C). Frequencies of subjects&#39;&#160;visitation to the selected three sites were also recorded. For Site A and Site C, the majority of subjects visited this site one to three times per week. For subjects at Site B, their frequency of visitation was equally split between one to three times per week and more than three times per week.
Biophysical and psychological stress indicators. Biophysical measures of cortisol and &#945;-amylase levels were utilized to identify changes in levels of physiological stress. Changes in psychological stress were identified through the PSQ instrument.
Effects of Recreational Site Visitation on Cortisol Levels and &#945;-amylase Levels 
The first research question asked if there would be a difference in levels of cortisol and &#945;-amylase as a function of type of site (e.g., level of nature). A paired sample t-test resulted in a significant decrease in salivary cortisol after visiting Site A (natural setting) [t31 = 3.26, p &#60; .01, see Figure 2]. No significant changes in levels of cortisol were observed in Site B and C. When comparing subjects changes in their cortisol levels across all three sites, the results of the ANOVA test indicated that different sites (levels of nature) did not have an overall significant impact on subjects changes in cortisol levels [F(2,95) = 1.86, p = 0.16] with small effect sizes (0.01-0.04).
Measurement of changes in levels of stress (pre/post visitation) using levels of &#945;-amylase resulted in mixed findings among the three study locations. Following the use paired sample t-tests indicate significant increases in levels of &#945;-amylase after visiting Site C [t34 = 2.79, p &#60; .01. Statistical differences were not observed after visitation to Sites A or Site B. (See Figure 3). Analysis using ANOVA techniques indicated a main effect of location with different locations had a significant impact on subjects changes in &#945;-amylase levels [F(2,101)= 3.36, p &#60; 0.05]. Using Scheffe post-hoc analysis, &#945;-amylase levels were significantly higher after visiting Site C when compared with Site B. There was a lack of significant differences on levels of &#945;-amylase when comparing visitors to Site A and Site B, or between Site A and Site C. Effect sizes of (0.03-0.01) were found and determined to be small.
Effects of Recreational Site Visitation on Psychological Stress Levels 
Paired sample t-tests were applied to compare pre- and post-visit of levels of psychological stress among the three sites, respectively. As shown in Figure 4 and 5, after visitation to the three locations, significant decreases on the factors of demands and worry (p &#60; 0.01) were observed. No significant changes in the factor, tension, was observed for any of the three locations. (See Figure 6). Significant increases were reported at Sites A and Site B for the factor, joy. No significant change was observed for visitors to Site C (see Figure 7).
To determine whether which of the three site is more efficacious in reducing subjects levels of psychological stress, the result of ANOVA tests indicated significant differences among the three sites (p &#60; 0.01) with post-hoc analysis using the Scheffe&#39;s Method, reporting significant increases in levels of joy after visiting Site A, compared to visitors visiting Sites B and Site C. No differences were found in changes in levels of demands, worries, and tension (p = 0.27) across three locations.
In summary, visitors to Site A (most natural) reported significant decreased levels of cortisol; suggesting a reduction in biological stress levels. In addition, as measured by the PSQ, significant decreases in the psychological factors of demands and worries, and a significant increase in the level of joy were observed in visitors to Site A. Visitors to Site B (semi-natural) reported decreases in levels of demands and worries, and an increased level of joy. C (built environment) two decreases in level of demands and worries were reported. Interestingly, levels of &#945;-amylase increased significantly after visitation to Site C. Further research is warranted to examining the influence of potential factors, such as specific activity or social surrounding, particularly on the catalyst, &#945;-amylase.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59272/59272fig2.jpg" /> 
Figure 2: Changes in levels of cortisol after visiting three different sites. This figure shows subjects&#39;&#160;cortisol levels measured before and after visiting three different sites representing different levels of nature: Sites A, B, and C. Data are presented as mean &#177; SD in natural log scale. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59272/59272fig3.jpg" /> 
Figure 3: Changes in levels of amylase after visiting three different sites. This figure shows subjects amylase levels measured before and after visiting Sites A, B, and C. Data are presented as mean &#177; SD in natural log scale. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59272/59272fig4.jpg" /> 
Figure 4: Changes in perceived levels of demands after visiting three different sites. This figure shows subjects&#39;&#160;levels of demands decreased after visiting Sites A, B, and C. Data are presented as mean &#177; SD. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59272/59272fig5.jpg" /> 
Figure 5: Changes in perceived levels of worries after visiting three different sites. This figure shows subjects&#39;&#160;levels of worries decreased after visiting Sites A, B, and C. Data are presented as mean &#177; SD. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/59272/59272fig6.jpg" /> 
Figure 6: Changes in perceived levels of tension after visiting three different sites. This figure shows subjects&#39;&#160;levels of tension decreased after visiting Sites A, B, and C. Data are presented as mean &#177; SD. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/59272/59272fig7.jpg" /> 
Figure 7: Changes in perceived levels of joy after visiting three different sites. This figure shows subjects&#39;&#160;levels of joys increased after visiting Sites A, B, and C. Data are presented as mean &#177; SD. <a href="https://www.jove.com/files/ftp_upload/59272/59272fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

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, &#945;-amylase, and a psychological self-report instrument.&#160;

Visitation to natural environments has been linked to psychological stress reduction. Although most stress-related research has relied on self-report ...

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8.4K 조회수.  Illinois State University. 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. 

비디오: Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature

The Trier Social Stress Test Protocol for Inducing Psychological Stress

This article demonstrates a psychological stress protocol for use in a laboratory setting. Protocols that allow researchers to study the biological pathways of the stress response in health and disease are fundamental to the progress of research in stress and anxiety.1 Although numerous protocols exist for inducing stress response in the laboratory, many neglect to provide a naturalistic context or to incorporate aspects of social and psychological stress. Of psychological stress protocols, meta-analysis suggests that the Trier Social Stress Test (TSST) is the most useful and appropriate standardized protocol for studies of stress hormone reactivity.2 In the original description of the TSST, researchers sought to design and evaluate a procedure capable of inducing a reliable stress response in the majority of healthy volunteers.3 These researchers found elevations in heart rate, blood pressure and several endocrine stress markers in response to the TSST (a psychological stressor) compared to a saline injection (a physical stressor).3 Although the TSST has been modified to meet the needs of various research groups, it generally consists of a waiting period upon arrival, anticipatory speech preparation, speech performance, and verbal arithmetic performance periods, followed by one or more recovery periods. The TSST requires participants to prepare and deliver a speech, and verbally respond to a challenging arithmetic problem in the presence of a socially evaluative audience.3 Social evaluation and uncontrollability have been identified as key components of stress induction by the TSST.4 In use for over a decade, the goal of the TSST is to systematically induce a stress response in order to measure differences in reactivity, anxiety and activation of the hypothalamic-pituitary-adrenal (HPA) or sympathetic-adrenal-medullary (SAM) axis during the task.1 Researchers generally assess changes in self-reported anxiety, physiological measures (e.g. heart rate), and/or neuroendocrine indices (e.g. the stress hormone cortisol) in response to the TSST. Many investigators have adopted salivary sampling for stress markers such as cortisol and alpha-amylase (a marker of autonomic nervous system activation) as an alternative to blood sampling to reduce the confounding stress of blood-collection techniques. In addition to changes experienced by an individual completing the TSST, researchers can compare changes between different treatment groups (e.g. clinical versus healthy control samples) or the effectiveness of stress-reducing interventions.1

This article describes a protocol for inducing psychological stress in participants, which enables researchers to measure psychological, physiological and neuroendocrine responses to stress within single participants or between groups.

심리적 스트레스를 유발을위한 모금 사회 스트레스 테스트 프로토콜

This article demonstrates a psychological stress protocol for use in a laboratory setting.  Protocols that allow researchers to study the biological ...

연구

의학

Assessing the Effects of Music Listening on Psychobiological Stress in Daily Life

Music listening is associated with stress-reducing effects. However, most of the results on music listening and stress were gathered in experimental settings. As music listening is a popular activity of daily life, it is of utmost importance to study the effects of music listening on psychobiological stress in an everyday, daily-life setting. Here, a study protocol is presented that allows the assessment of associations between music listening and psychobiological stress in daily life by noninvasively measuring salivary cortisol (as a marker of the Hypothalamic-Pituitary-Adrenal (HPA) axis) and salivary alpha-amylase (as a marker of the Autonomic Nervous System (ANS)). The protocol includes advice on the study design (e.g., sampling protocol), the materials and methods (e.g., the assessment of psychobiological stress in daily life, the assessment of music listening, and the manual), the selection of participants (e.g., the approval of the institutional review board and inclusion criteria), and the statistical analyses (e.g., the multilevel approach). The representative results provide evidence for a stress-reducing effect of music listening in daily life. Particularly, specific reasons for listening to music (especially relaxation), as well as the presence of others while doing so, increase this stress-reducing effect. At the same time, music listening in daily life differentially affects the HPA axis and ANS functioning, thus emphasizing the need for a multi-dimensional assessment of stress in daily life.

A study protocol is presented on how to assess associations between music listening and psychobiological stress (as measured by subjective stress levels, salivary cortisol, and salivary alpha-amylase) in daily life. Advice on study design, materials, methods, selection of participants, and statistical handling is provided. Representative results are presented and discussed.

Music listening is associated with stress-reducing effects. However, most of the results on music listening and stress were gathered in experimental ...

행동분석학

A Community-based Stress Management Program: Using Wearable Devices to Assess Whole Body Physiological Responses in Non-laboratory Settings

A pragmatic breath-based intervention to benefit human performance and stress management is timely and valuable to individuals seeking holistic approaches for emotional regulation and optimizing compensatory reserve mechanisms. This protocol is designed to not only teach mind-body awareness but also to provide feedback utilizing physiological data and survey results. The primary findings of this study showed that heart coherence and alpha variables were significantly correlated after four weeks of the breath-based meditation stress protocol. Meditation and rhythmic breathing produced significant increases in alpha brain activity. These brain physiological responses conformed to the Pleth Variability Index (PVI) changes, suggesting the ability of the human body to enter into a meditative state and effectively manage stress. When assessed after four weeks of daily practicing the techniques employed in the stress management protocol, based on the Five Facet Mindfulness Questionnaire, subjects improved in applying mindfulness skills. The overall mindfulness score, Pleth variability index (PVI), and perfusion index (PI) increased after the 4-week intervention period. Results from electroencephalography (brain waves) were consistent with a meditative state during the post-study follow-up session. This provides evidence that wearable devices are feasible for data collection during a breath-based stress management intervention. This protocol can be easily and efficiently implemented into any study design in which physiological data are desired in a non-laboratory-based setting.

Stress is an unavoidable and persistent component of life and holistic approaches for its management are being considered. A standardized methodology was created to demonstrate the feasibility of a breath-based stress management protocol that can be used with commercially available portable technology.

커뮤니티 기반 스트레스 관리 프로그램: 전체를 평가 하기 위해 착용 할 수 있는 장치를 사용 하 여 바디 비 실험실 설정에서 생리 적인 응답

A pragmatic breath-based intervention to benefit human performance and stress management is timely and valuable to individuals seeking holistic approaches ...

A Modified Trier Social Stress Test for Vulnerable Mexican American Adolescents

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the biological output produced by the Hypothalamic-Pituitary-Adrenal (HPA) axis in humans. While studies have explored how modifications to the TSST influence stress responsivity, few studies have created a modified TSST appropriate for vulnerable youth that elicits a significant cortisol stress response. Thus, the current study sought to modify or adjust the TSST in a culturally sensitive way for a vulnerable sample of 14 year-old adolescents. The present study took place within the context of a longitudinal birth cohort study of Mexican American families in California called the Center for the Health Assessment of Mothers and Children of Salinas (CHAMACOS). The CHAMACOS sample was optimal to test the effectiveness of a modified culturally appropriate TSST, as it is comprised of Mexican American youth, who are often excluded from research. These youths also have experienced significant early life adversity. Example modifications included timed prompts, alternative math tasks, use of same-ethnicity peers as confederates, debriefing immediately after the conclusion of the TSST, and using an unknown youth examiner to administer the debrief. Saliva samples were collected at baseline (after a resting phase), and then again at 15, 30, and 45 min post-TSST onset to assess cortisol concentration. A pilot study of 50 participants (50% female) have been analyzed for cortisol reaction to the TSST. Results confirmed that this modified version of the TSST was successful at eliciting a significant cortisol reaction, with a wide range of variability likely due to individual differences. Goals for modifications and ethnicity considerations are discussed. This study provides the foundation for future research to utilize a modified TSST with vulnerable youth.

Here, we present a protocol that provoked cortisol reactivity in a vulnerable adolescent Mexican American sample utilizing a modified version of the Trier Social Stress Test (TSST). Saliva samples were collected at baseline, 15, 30, and 45 min post-TSST onset. Future research could utilize this modified TSST with vulnerable youth.

The Trier Social Stress Test (TSST) is a well validated and widely used social stressor that has been shown to induce a 2-4 fold increase in cortisol, the ...

발생학

Virtual Reality Experiments with Physiological Measures

Virtual reality (VR) experiments are increasingly employed because of their internal and external validity compared to real-world observation and laboratory experiments, respectively. VR is especially useful for geographic visualizations and investigations of spatial behavior. In spatial behavior research, VR provides a platform for studying the relationship between navigation and physiological measures (e.g., skin conductance, heart rate, blood pressure). Specifically, physiological measures allow researchers to address novel questions and constrain previous theories of spatial abilities, strategies, and performance. For example, individual differences in navigation performance may be explained by the extent to which changes in arousal mediate the effects of task difficulty. However, the complexities in the design and implementation of VR experiments can distract experimenters from their primary research goals and introduce irregularities in data collection and analysis. To address these challenges, the Experiments in Virtual Environments (EVE) framework includes standardized modules such as participant training with the control interface, data collection using questionnaires, the synchronization of physiological measurements, and data storage. EVE also provides the necessary infrastructure for data management, visualization, and evaluation. The present paper describes a protocol that employs the EVE framework to conduct navigation experiments in VR with physiological sensors. The protocol lists the steps necessary for recruiting participants, attaching the physiological sensors, administering the experiment using EVE, and assessing the collected data with EVE evaluation tools. Overall, this protocol will facilitate future research by streamlining the design and implementation of VR experiments with physiological sensors.

Virtual reality (VR) experiments can be difficult to implement and require meticulous planning. This protocol describes a method for the design and implementation of VR experiments that collect physiological data from human participants. The Experiments in Virtual Environments (EVE) framework is employed to accelerate this process.

가상 현실 생리 측정 실험

Virtual reality (VR) experiments are increasingly employed because of their internal and external validity compared to real-world observation and ...

Enactive Phenomenological Approach to the Trier Social Stress Test: A Mixed Methods Point of View

The single Trier Social Stress Test (TSST) and the TSST for groups (TSST-G) are the most used protocols to experimentally induce psychosocial stress. These tests are based on uncontrollability and social-evaluative threat, inducing psychological and physiological consequences (e.g., anxiety, emotional states, salivary cortisol increases). Many quantitative experimental studies have investigated these stress inducers and these consequences. But, as far as we know, this study is the first to provide a qualitative analysis to access the participants' voices so as to understand the dynamics of their experience throughout the TSST and the TSST-G. This paper outlines a mixed methods approach to the TSST. This approach can help to maximize the information that can be gained from the TSST, which researchers often use without looking more closely at what is qualitatively happening psychologically for participants during the stressor itself. In this way, this protocol is an example of mixed methods, showing the added value of using the enactive phenomenological approach to analyze experimental protocols more deeply. This kind of mixed methods is helpful to access the experience, to understand the actor's point of view, and to analyze in-depth the dynamics of cognitive processes like intentions, perceptions, enacted knowledge, and emotion. The discussion section shows the different uses of a mixed methods protocol, exploiting the enactive phenomenological approach to analyze a protocol or to give a cross vision of the same research subject. This section deals with different existing applications, pointing out some critical steps in this mixed methods approach.

Here, we present an original mixed method combining a quantitative and a qualitative approach to examine accurately participants&#39; experiences during the Trier Social Stress Test and the Trier Social Stress Test for groups.

트리에 사회 스트레스 테스트에 enactive 현상 학적 접근: 혼합된 방법의 관점

The single Trier Social Stress Test (TSST) and the TSST for groups (TSST-G) are the most used protocols to experimentally induce psychosocial stress. ...

Evaluation of Commercial-Off-The-Shelf Wrist Wearables to Estimate Stress on Students

Wearable commercial-off-the-shelf (COTS) devices have become popular during the last years to monitor sports activities, primarily among young people. These devices include sensors to gather data on physiological signals such as heart rate, skin temperature or galvanic skin response. By applying data analytics techniques to these kinds of signals, it is possible to obtain estimations of higher-level aspects of human behavior. In the literature, there are several works describing the use of physiological data collected using clinical devices to obtain information on sleep patterns or stress. However, it is still an open question whether data captured using COTS wrist wearables is sufficient to characterize the learners' psychological state in educational settings. This paper discusses a protocol to evaluate stress estimation from data obtained using COTS wrist wearables. The protocol is carried out in two phases. The first stage consists of a controlled laboratory experiment, where a mobile app is used to induce different stress levels in a student by means of a relaxing video, a Stroop Color and Word test, a Paced Auditory Serial Addition test, and a hyperventilation test. The second phase is carried out in the classroom, where stress is analyzed while performing several academic activities, namely attending to theoretical lectures, doing exercises and other individual activities, and taking short tests and exams. In both cases, both quantitative data obtained from COTS wrist wearables and qualitative data gathered by means of questionnaires are considered. This protocol involves a simple and consistent method with a stress induction app and questionnaires, requiring a limited participation of support staff.

A protocol to evaluate solutions based on commercial-off-the-shelf (COTS) wrist wearables to estimate stress in students is proposed. The protocol is carried out in two phases, an initial laboratory-based stress induction test, and a monitoring stage taking place in the classroom while the student is performing academic activities.

상업-오프-더-선반 손목 Wearables 추정 학생에 대 한 스트레스의 평가

Wearable commercial-off-the-shelf (COTS) devices have become popular during the last years to monitor sports activities, primarily among young people. ...

공학

An Application for Pairing with Wearable Devices to Monitor Personal Health Status

The current protocol aims to showcase the technology integration, providing a detailed description of adopting the HealthCloud app, developed by the Healthy Landscape and Healthy People Lab, National Taiwan University (HLHP-NTU), on smartphones and smartwatches to collect data on users' real-time psychological and physiological responses and environmental information. A flexible and integrated research method was proposed because it can be difficult to measure multi-dimensional aspects of personal data in on-site studies in landscape and outdoor recreation research. An on-site study conducted in 2020 at the National Taiwan University campus was used as an application example. A dataset of 385 participants was used after excluding invalid samples. During the experiment, participants were asked to walk around campus for 30 min when their heart rate and psychological-scale items were measured, together with several environmental metrics. This work aimed to provide a possible solution to help on-site studies track real-time human responses that match ambient factors. Due to the app's flexibility, its use on wearable devices shows excellent potential for multidisciplinary research studies.

The present protocol introduces a non-commercial self-developed application for collecting real-time on-site data, including psychological scales, GPS location, heart rate, and blood-oxygen saturation level, as well as the application's operating procedures. An empirical study conducted in Taiwan in 2020 was used as an application example.

개인 건강 상태를 모니터링하기 위해 웨어러블 장치와 페어링하는 응용 프로그램

The current protocol aims to showcase the technology integration, providing a detailed description of adopting the HealthCloud app, developed by the ...

환경과학

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

생체 자기 제어를 사용 Psychophysiological 스트레스 평가

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

생물학

Thermal Imaging to Study Stress Non-invasively in Unrestrained Birds

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood flow that results in a net influx of blood to key organs and an increase in core temperature. This stress-induced hyperthermia is used to assess stress. However, measuring core temperature is invasive. As blood flow is redirected to the core, the periphery of the body can cool. This paper describes a protocol where peripheral body temperature is measured non-invasively in wild blue tits (Cyanistes caeruleus) using infrared thermography. In the field we created a set-up bringing the birds to an ideal position in front of the camera by using a baited box. The camera takes a short thermal video recording of the undisturbed bird before applying a mild stressor (closing the box and therefore capturing the bird), and the bird&#8217;s response to being trapped is recorded. The bare skin of the eye-region is the warmest area in the image. This allows an automated extraction of the maximum eye-region temperature from each image frame, followed by further steps of manual data filtering removing the most common sources of errors (motion blur, blinking). This protocol provides a time series of eye-region temperature with a fine temporal resolution that allows us to study the dynamics of the stress response non-invasively. Further work needs to demonstrate the usefulness of the method to assess stress, for instance to investigate whether eye-region temperature response is proportional to the strength of the stressor. If this can be confirmed, it will provide a valuable alternative method of stress assessment in animals and will be useful to a wide range of researchers from ecologists, conservation biologists, physiologists to animal welfare researchers.

There is a need for a non-invasive assessment of stress. This paper describes a simple protocol using thermal imaging to detect a significant response in eye-region temperature in wild blue tits to a mild acute stressor.

열 화상 비 침습적 무제한 조류의 스트레스를 연구

Stress, a central concept in biology, describes a suite of emergency responses to challenges. Among other responses, stress leads to a change in blood ...