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. ...

<ol>
	<li>Hansmann, R., Hug, S., Seeland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+and+stress+relief+through+physical+activities+in+forests+and+parks.">Restoration and stress relief through physical activities in forests and parks.</a> Urban Forestry and Urban Greening. 6, 213-225 (2007).</li><li>Krantz, D. S., McCeney, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+psychological+and+social+factors+on+organic+disease:+A+critical+assessment+of+research+on+coronary+heart+disease.">Effects of psychological and social factors on organic disease: A critical assessment of research on coronary heart disease.</a> Annual Review of Psychology. 53, 341-369 (2002).</li><li>Ward Thompson, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=More+green+space+is+linked+to+less+stress+in+deprived+communities:+Evidence+from+salivary+cortisol+patterns.">More green space is linked to less stress in deprived communities: Evidence from salivary cortisol patterns.</a> Landscape and Urban Planning. 105, 221-229 (2012).</li><li>Haluza, D., Schonbauer, R., Cervinka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+perspectives+for+public+health:+A+narrative+on+the+physiological+effects+of+experiencing+outdoor+nature.">Green perspectives for public health: A narrative on the physiological effects of experiencing outdoor nature.</a> International Journal of Environmental Research and Public Health. 11, 5445-5461 (2014).</li><li>Korpela, K. M., Ylen, M., Tyrv&#228;inen, L., Silvennomen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determinants+of+restorative+experiences+in+everyday+favorite+places.">Determinants of restorative experiences in everyday favorite places.</a> Health and Place. 14, 636-652 (2008).</li><li>Mantler, A., Logan, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+environments+and+mental+health.">Natural environments and mental health.</a> Advances in Integrative Medicine. 2, 5-12 (2015).</li><li>Mayer, F. S., McPherson-Frantz, C., Bruehlman-Senecal, E., Dolliver, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+is+nature+beneficial?+the+role+of+connectedness+to+nature.">Why is nature beneficial? the role of connectedness to nature.</a> Environment and Behavior. 41, 307-643 (2009).</li><li>Pretty, J., Peacock, J., Sellens, M., Griffin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mental+and+physical+health+outcomes+of+green+exercise.">The mental and physical health outcomes of green exercise.</a> International Journal of Environal Health Research. 15, 319-337 (2005).</li><li>Ulrich, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+recovery+during+exposure+to+natural+and+urban+environments.">Stress recovery during exposure to natural and urban environments.</a> Journal of Environmental Psychology. 11, 201-230 (1991).</li><li>Kaplan, S., Talbot, J. F., Altman, I., Wohlwill, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psychological+Benefits+of+a+Wilderness+Experience.">Psychological Benefits of a Wilderness Experience.</a> Behavior and the Natural Environment. , 163-203 (1983).</li><li>Salmon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+physical+exercise+on+anxiety,+depression,+and+sensitivity+to+stress:+A+unifying+theory.">Effects of physical exercise on anxiety, depression, and sensitivity to stress: A unifying theory.</a> Clinical Psychology Review. 21, 33-61 (2001).</li><li>Focht, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+walks+in+outdoor+and+laboratory+environments:+Effects+on+affective+responses,+enjoyment,+and+intentions+to+walk+for+exercise.">Brief walks in outdoor and laboratory environments: Effects on affective responses, enjoyment, and intentions to walk for exercise.</a> Research Quarterly for Exercise and Sport. 80, 611-620 (2009).</li><li>Fliege, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Perceived+Stress+Questionnaire+(PSQ)+reconsidered:+Validation+and+reference+values+from+different+clinical+and+healthy+adult+samples.">The Perceived Stress Questionnaire (PSQ) reconsidered: Validation and reference values from different clinical and healthy adult samples.</a> Psychosomatic Medicine. 67, 78-88 (2005).</li><li>Kirschbaum, C., Hellhammer, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+cortisol+in+psychoneuronendocrine+research:+Recent+developments+and+applications.">Salivary cortisol in psychoneuronendocrine research: Recent developments and applications.</a> Psychoneuroendocrinology. 19, 313-333 (1994).</li><li>Gallacher, D. V., Petersen, O. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-secretion+coupling+in+mammalian+salivary+glands.">Stimulus-secretion coupling in mammalian salivary glands.</a> International Reviews in Physiology. 28, 1-52 (1983).</li><li>Slosnik, R. T., Chatterton, R. T., Swisher, T., Par, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+attentional+inhibition+by+norepinephrine+and+cortisol+after+psychological+stress.">Modulation of attentional inhibition by norepinephrine and cortisol after psychological stress.</a> International Journal of Psychophysiology. 36, 59-68 (2000).</li><li>Nater, U. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-induced+changes+in+human+salivary+alpha-amylase+activity-associations+with+adrenergic+activity.">Stress-induced changes in human salivary alpha-amylase activity-associations with adrenergic activity.</a> Psychoneuroendocrinology. 31 (1), 49-58 (2006).</li><li>Takai, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+psychological+stress+on+the+salivary+cortisol+and+amylase+levels+in+healthy+young+adults.">Effect of psychological stress on the salivary cortisol and amylase levels in healthy young adults.</a> Archives of Oral Biology. 49 (12), 963-968 (2004).</li><li>Shirtcliff, E. A., Granger, D. A., Schwatz, E., Curran, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+salivary+biomarkers+in+biobehavioral+research:+Cotton+based+sample+collection+methods+can+interfere+with+salivary+immunoassay+results.">Use of salivary biomarkers in biobehavioral research: Cotton based sample collection methods can interfere with salivary immunoassay results.</a> Psychoneuroendocrinology. 26, 165-173 (2001).</li><li>Nater, U. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+salivary+alpha-amylase+reactivity+in+a+psychosocial+stress+paradigm.">Human salivary alpha-amylase reactivity in a psychosocial stress paradigm.</a> Journal of Psychophysiology. 55 (3), 333-342 (2005).</li><li>Granger, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+salivary+biomarkers+into+developmental+and+behaviorally-oriented+research:+Problems+and+solutions+for+collecting+specimens.">Integration of salivary biomarkers into developmental and behaviorally-oriented research: Problems and solutions for collecting specimens.</a> Physiology and Behavior. 92, 583-590 (2007).</li><li>Frumkin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+toxicity:+Human+health+and+the+natural+environment.">Beyond toxicity: Human health and the natural environment.</a> American Journal of Preventive Medicine. 20, 234-240 (2001).</li><li>Hartig, T., Mitchell, R., de Vries, S., Frumkin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nature+and+health.">Nature and health.</a> Annual Review of Public Health. 35, 207-228 (2014).</li><li>Gidlow, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Where+to+put+your+best+foot+forward:+Psycho-physiological+responses+to+walking+in+natural+and+urban+environments.">Where to put your best foot forward: Psycho-physiological responses to walking in natural and urban environments.</a> Journal of Environmental Psychology. 45, 22-29 (2016).</li><li>Ewert, A., Chang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levels+of+nature+and+stress+response.">Levels of nature and stress response.</a> Behavioral Sciences. 8 (5), 49 (2018).</li><li>Wyles, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+some+natural+environments+more+psychologically+beneficial+than+others?+The+importance+of+type+and+quality+on+connectedness+to+nature+and+psychological+restoration.">Are some natural environments more psychologically beneficial than others? The importance of type and quality on connectedness to nature and psychological restoration.</a> Environment and Behavior. 51 (2), 111-143 (2019).</li></ol>

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 numbe...

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 border="0" cellpadding="0" cellspacing="0" style="width:1240px;" width="1240">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="53">
			<td height="53" style="height:54px;width:271px;">Cortisol Enzyme Immunoassay Kit</td>
			<td style="width:173px;">DetectX</td>
			<td style="width:119px;">K003-H1</td>
			<td style="width:677px;">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 height="53">
			<td height="53" style="height:54px;width:271px;">Cryogenic Labels for Cryogenic Storage</td>
			<td style="width:173px;">Fisherbrand</td>
			<td style="width:119px;">5-910-A</td>
			<td style="width:677px;">Unique adhesive withstands extreme temperature</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:271px;">Liquid Amylase (CNPG3) Reagent Set</td>
			<td style="width:173px;">Pointe Scientific</td>
			<td style="width:119px;">A7564</td>
			<td>For the quantitative kinetic determination of &#945;-amylase activity in human serum.</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:271px;">Round Bottom 2mL Polypropylene Tubes with External Thread Cap</td>
			<td style="width:173px;">Greiner Bio-One</td>
			<td style="width:119px;">07-000-257</td>
			<td style="width:677px;">2.0 ml U-BTM Cryo.s self standing polypropylene sterilized</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:54px;width:271px;">Synergy Multi-Mode Microplate Reader</td>
			<td style="width:173px;">BioTek</td>
			<td></td>
			<td style="width:677px;">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 on...

Watch this Scientific Journal Video about Measuring Biophysical and Psychological Stress Levels Following Visitation to Three Locations with Differing Levels of Nature at JoVE.com

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 ...

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.

measuring-biophysical-psychological-stress-levels-following

Research

JoVE Journal

Behavior

8.4K visualizações.  Illinois State University. Aqui apresentamos um protocolo, usado para examinar os efeitos sobre os níveis de estresse biofísico e psicológico após uma visitação a três locais com diferentes níveis de natureza. Estamos interessados em saber se um site com níveis mais altos de naturalidade será mais eficaz em ajudar os participantes a reduzir seus níveis de estresse. Além do uso do questionário de autorre relato, a principal vantagem do uso dessa técnica é que analisar os níveis de cortisol e amilase cole...