Do Terpenes Play a Role in the Stress-reducing Effects of a Forest Bathing Intervention?
Phase: NA
Conditions: Monoterpene Exposure During a Forest Bathing Intervention
Sponsor: University of Washington

Do Terpenes Play a Role in the Stress-reducing Effects of a Forest Bathing Intervention? 
NCT05316597 
May 7, 2024  
 
 

1. Objectives 
Aim 1. Assess whether VOC inhalation regulates stress reduction and affective outcomes of the terpenes-
on vs. terpenes-off sessions. 
Primary physiological outcome: increases in the HF (ms2) component of HRV. 
Secondary outcomes: decreases in blood pressure, heart rate, self-reported stress, negative affect, and 
levels of inflammatory cytokines (IL-6, TNF-alpha, CRP) and cortisol in serum, and increases in positive 
affect, measured via mobile physiology equipment and blood using standard clinical methods. 
Hypothesis: VOC inhalation will regulate increases in the HF (ms2) component of HRV and decreases in 
blood pressure, heart rate, self-reported stress, and levels of inflammatory cytokines in serum as 
secondary outcomes. 
Aim 1a. Assess the degree of association of absorbed dose of seven forest-derived VOCs in serum (i.e., α- 
pinene, β-pinene, β-myrcene, ∆ 3- carene, limonene, β- carophyllene, α-humulene) with these outcomes. 
Hypothesis: Absorbed dose of seven forest-derived VOCs in serum will be associated with stress 
reduction and affective outcomes.  
 
2. Design 
Potential participants were screened via a phone call and excluded if they were pregnant, smoking, and/or 
had a current or prior diagnosis of neurologic, hypertensive, psychiatric, respiratory disorder, or 
anosmia/hyposmia. Participants were also excluded if they were prescribed a short list of prescription 
medications known to influence terpene metabolic pathways and short-term inflammatory biomarkers 
including beta-blockers, antibiotics, statins, hypertension medications, steroid medications, and diabetes 
control medications. Eligible participants were asked to avoid certain foods, beverages, cleaning products, 
and supplements that contain terpene compounds in the 24 hours leading up to their forest sitting 
experience. 
Additionally, the clinically validated University of Pennsylvania Smell Identification Test 
(UPSIT) (Sensonics International, Haddon Heights, NJ) was administered to determine whether 

participants had anosmia/hyposmia. The UPSIT is a 40-item, self-administered “scratch-and-sniff” test 
that uses microencapsulated odorants that are released by scratching designated spaces on a paper test 
booklet. Summed scores were used to evaluate olfactory function and identify/exclude participants with 
undiagnosed smell loss or total anosmia (summed UPSIT score ≤18). 
Following enrollment, participants were randomly assigned to a sequence of two sessions using a 
computer-randomized order created by an investigator who was not a member of the field team. To 
control participants inhalation exposures to terpenes in the forest air, participants wore a powered air-
purifying respirators (PAPRs; 3M™ Versaflo™ Powered Air Purifying Respirator, TR-800-PSK/94248; 
3M, St. Paul, MN). In one session, the PAPR mask was fitted with a particle-only filter (3M™ Versaflo™ 
High-Efficiency Filter, TR-6710N-5) to allow for the inhalation of forest terpenes (“terpenes-on”). In the 
other session, the mask was fitted with a charcoal filter (3M™ Versaflo™ Organic Vapor/HEPA 
Cartridge, TR-6510N) that removed particles, terpenes, and other BVOCs from the breathing zone 
(“terpenes-off”). Equipment labels were covered and filters were labeled “A” (later revealed to be 
“terpenes-on”) or “B” (later revealed to be “terpenes-off”) to blind the study field team and participants to 
treatment assignment. A washout period of at least eight days between sessions was included to reduce 
carryover effects (Sibbald & Roberts, 1998). 
Forest-sitting sessions took place at the UW Charles L. Pack Experimental Forest located near 
Eatonville, Washington (“Pack Forest”) during June to September 2022 and 2023 to avoid rain, snow, and 
cold weather. The sessions were 60 minutes each and took place between 10:00 AM and 4:00 PM in a 
stand of old- and second-growth Douglas Fir, Western Red Cedar, and other conifers accessible by a 10-
minute drive on an unpaved single-lane road from the Pack Forest Conference Center. Participants were 
seated in a comfortable chair, but the session was otherwise unscripted. 
 
3. Methods 
We enrolled 43 adult participants between July 12, 2022, and September 21, 2023. Our target sample size 
of 40 participants was based on the estimated numbers needed to detect minimum differences in the study 

outcomes in the contrasting conditions for the same participant, based on the minimums in the ranges of 
Hedges’ g values from prior studies and standard population-level intra-individual differences. 
Participants were recruited from the Tacoma and Seattle, WA area using physical flyers placed in 
community centers, libraries, and local universities, and electronic listings on Craigslist and the 
University of Washington (UW) Institute of Translational Health Sciences study recruitment site. 
Potential participants were screened via phone call and excluded if they were pregnant, smoking, not 
fluent in English, and/or had a current or prior diagnosis of neurologic, hypertensive, psychiatric, or 
respiratory disorder, or anosmia/hyposmia. Participants were also excluded if they were prescribed a short 
list of prescription medications known to influence terpene metabolic pathways and/or short-term 
inflammatory biomarkers including beta-blockers, antibiotics, statins, hypertension medications, steroid 
medications, and diabetes control medications or if they were unable to walk for 15-20 min on unsteady 
ground. Eligible participants were asked to avoid certain consumer products, foods, beverages, cleaning 
products, alcohol, marijuana, e-cigarettes, and supplements that contain terpenes in the 24 hours leading 
up to their forest-sitting experience. 
Participants’ usual nature contact was assessed using measures from Bratman et al. (2024) and 
Bratman et al. (2021), based on operationalizations from other nature exposure papers (Shanahan et al., 
2016; White et al., 2019). Average nature contact frequency was assessed by presenting asking 
participants with the following question: “About how often do you usually visit or pass through outdoor 
natural areas for any reason? This includes, for example, walking, biking, or recreating outside in local, 
regional, or national parks, at the beach, beside or within lakes, creeks, or the ocean, gardening or tending 
to plants, camping, fishing, reading or walking outside next to trees, engaging in yard work with natural 
elements, etc…” Participants selected an answer from following options: “Never”, “Once a year”, “Once 
every three months”, “Once a month”, “2-3 times a month”, “Once a week”, “2-3 days a week”, “4-5 days 
a week”, or “6-7 days a week” (Bratman et al., 2024). Average nature contact duration was measured by 
presenting participants with the following question: “Over the last month, approximately how many 
HOURS PER WEEK do you consider yourself to have interacted with nature? This includes, for example, 

walking, biking, or recreating outside in local, regional, or national parks, at the beach, beside or within 
lakes, creeks, or the ocean, gardening or tending to plants, camping, fishing, reading or walking outside 
next to trees, engaging in yard work with natural elements, etc...” (Bratman et al., 2021). Participants 
entered their response in a provided text box.  
Nature relatedness was measured during baseline measurements using the short-form nature 
relatedness scale (NR-6; Nisbet & Zelenski, 2013). The NR-6 consists of 6 items designed to capture how 
people view their relationship with nature (e.g., “My relationship to nature is an important part of who I 
am”). Each item is rated on a 5-point scale, ranging from 1 (“Disagree strongly”) to 5 (“Agree strongly”) 
with a total score calculated by averaging all six items. Cronbach’s α was used to measure how closely 
correlated related scale items were as a group. In the current study, NR-6 showed good reliability 
(Cronbach’s α = .76).   
HF HRV was measured in milliseconds squared (ms2) using a portable, continuous 
electrocardiogram sensor (EcgMove4; Movisens®, Karlsruhe, Germany) worn directly on the chest with 
adhesive electrodes. We applied a natural logarithmic transformation to 5-minute averages of HF HRV 
(‘ln-HF HRV') following Shaffer & Ginsberg (2017). Additionally, HF HRV values were winsorized. 
Systolic and diastolic blood pressure were measured with an automated cuff monitor (GE 
Healthcare CARESCAPE V100 Vital Signs Monitor; GE Healthcare, Chicago, IL) in millimeters of 
mercury (mmHg). HR was also measured with the automated cuff monitor in beats per minute (BPM). 
SCLs were measured in microsiemens (µS) using a portable, continuous electrodermal sensor 
(EdaMove4; Movisens®, Karlsruhe, Germany) worn on a wristband and attached to the palm with 
adhesive electrodes. 
General levels of positive and negative affect were assessed using the 20-item Positive and 
Negative Affect Schedule (PANAS; Watson et al., 1988) during baseline measurements and state levels 
were assessed during the forest-sitting sessions using the 10-item, shortened PANAS (I-PANAS-SF; 
Thompson, 2007). The PANAS is designed to assess affect over the past month and includes 10 items on 
positive affect (e.g., “Interested,” “Active”) and 10 on negative affect (“Distressed,” “Irritable”). Each 

item is rated on a 5-point scale, ranging from 1 (“very slightly or not at all”) to 5 (“extremely”) with sum 
positive and negative affect scores calculated. In the current study, the PANAS showed good reliability 
for positive affect (α = .88) and negative affect (α = .85). On the I-PANAS-SF, 5 items assess positive 
affect (e.g., “Inspired”, “Alert”), and five assess negative affect (“Upset”, “Hostile”) at the present 
moment. Each item is rated on a five-point scale, ranging from 1 (“very slightly or not at all”) to 5 
(“extremely”) from which sum positive and negative affect scores were calculated. In the current study, I-
PANAS-SF showed good reliability for positive affect (Cronbach’s α = .86) and lower reliability for 
negative affect (Cronbach’s α = .40). 
Baseline perceived stress was assessed using the four-item version of the Perceived Stress Scale 
(PSS-4) designed to assess perceptions of stress over the last month (e.g., “In the last month, how often 
have you felt that you were unable to control the important things in your life?”; Cohen et al., 1983). Each 
item was rated on a 5-point scale, ranging from 0 (“never”) to 4 (“very often”) and a sum score was 
calculated. In the current study, PSS-4 showed good reliability (Cronbach’s α = .78). State-level stress 
during the forest sessions was measured with one item. Participants were asked to rate to what extent they 
felt stressed at the moment on a 5-point scale, ranging from 1 (“very slightly or not at all”) to 5 
(“extremely”). 
Different aspects of the subjective experience of the forest setting were assessed following each 
session using a combination of questions about the sensory experience and the perceived restorativeness 
of the forest. Perceived restorativeness was assessed using the adapted 11-item Perceived Restorativeness 
Scale (PRS-11) which asks participants to rate the degree to which they agreed with statements such as 
“Places like that are fascinating” using an 11-point scale, ranging from 0 (“not at all”) to 10 
(“completely”; Pasini et al., 2014),  Additionally, participants were asked to rate the pleasantness of their 
sensory experience of the forest for sight, smell, and visual experience on a 10-point scale ranging from 1 
(“not pleasant”) to 10 (“extremely pleasant”). Participants were also provided an open-ended text box to 
describe further thoughts or feelings they had about the sensory experience of the forest. 

A trained phlebotomist or registered nurse collected approximately 5 mL of blood via venipuncture 
before and after each session using BD Vacutainer™ Push Button Blood Collection Sets into 6 ml BD 
Vacutainer™ Venous Blood Collection Tubes (BD, Franklin Lakes, NJ). On the day of collection, 
samples were stored at room temperature for 30 min to allow clotting, stored at  4°C, then centrifuged at 
3,000 rpm for 10 min . Serum aliquots were separated into 2 mL Fisherbrand™ Externally and Internally 
Threaded Cryogenic Storage Vials (Thermo Fisher Scientific, Waltham, MA) and stored at -80°C before 
being transported on dry ice to the Centers for Disease Control and Prevention (CDC) Tobacco and 
Volatiles Branch (Atlanta, GA) and the UW Center for Studies in Demography and Ecology 
Biodemography Laboratory (Seattle, WA) for analysis.  
Acute cytokine response was assessed using a custom three-plex enzyme immunoassay microarray 
(Quansys Biosciences, Logan, UT, part number 107749GR) to measure levels of CRP, TNF-α, IL-6, and 
serum cortisol. Chemiluminescence was quantified using a Quansys Q-view Imager LS. An 8-point, 5-
parameter standard curve was used to estimate cytokine concentrations and assay limits of detection (Q-
view software, Quansys Biosciences, Logan, UT).  
A competitive microplate enzyme immunoassay, previously validated for use with plasma 
(Munro & Stabenfeldt, 1984), was adapted to measure cortisol in serum extracts using a purified 
polyclonal anti-cortisol antibody, R4866 (provided by C. Munro, UC Davis), and cortisol reference 
calibrators (Steraloids, catalog number Q3880). This assay has been used successfully with saliva, urine, 
hair, and dried blood spot samples (Doyle et al., 2019; Heller et al., 2018; Konishi et al., 2012; Trumble et 
al., 2010). The anti-cortisol antibody cross-reacts 100% with cortisol, 10% with prednisolone, 6% with 
prednisone, 6% with 11-deoxycortisol, 5% with cortisone, and less than 1% with all other steroids (data 
provided by C. Munro). Color reactions were quantified at 405 nm (test) and 570 nm (reference) using a 
Synergy HT microplate reader (Bio Tek Instruments, Inc., Winooski, VT). A 5-parameter standard curve 
was used to estimate cortisol concentrations (Gen5, Bio Tek Instruments Inc., Winooski, VT). For both 
acute cytokine and cortisol response analyses, all samples, standards, and controls were assayed in 
duplicate wells.  

Concentrations of α-pinene, β-caryophyllene, β-myrcene, β-pinene, Δ-3-carene, and limonene 
were measured in serum using stable isotope dilution with headspace solid-phase microextraction (SPME) 
and gas chromatography-tandem mass spectrometry (HS-SPME GC-MS/MS) following Silva et al. 
(2020). Methods and results are detailed in Riederer et al. (in preparation). Briefly, samples were thawed, 
mixed using a hematology mixer (Fisher Scientific Inc., Pittsburgh, PA), and a 0.50 mL aliquot dispensed 
into a 10 mL solid phase microextraction vial. Purge-and-trap grade methanol was used to dilute pure 
chemicals into the primary standard and internal standard (ISTD) stock solutions. A 40 μL spike of the 
ISTD solution was added the sample, which was then crimp-sealed and mixed with a vortexer (S/P Multi-
Tube Vortexer, Baxter Diagnostics Inc., Minster, OH) for 5 min. Vials were placed on a Peltier-cooled 
sample tray (15°C) on an Agilent 7890 GC (Agilent, Santa Clara, CA) coupled to an Agilent 7010 triple-
quadrupole MS (Agilent, Santa Clara, CA) with a mounted CombiPAL autosampler and extra PAL 
autosampler arm (CTC Analytics AG, Zwingen, Switzerland). Samples were analyzed in positive ion 
electron-impact ionization with multiple reaction monitoring modes. Blank and quality control samples 
were also prepared with a 40 μL ISTD spike. Standards prepared following an identical method were 
included with each analytical run. Laboratory blanks consisted of VOC-free water in sealed ampoules 
opened on the day of analysis. We also collected 7 field blanks on random days comprised of VOC-free 
water drawn through serum tubes and handled identically to the serum samples.  
Terpenes were measured in air at the forest site while participants were present using a method 
adapted from NIOSH Method 1552 (National Institute for Occupational Safety and Health, 1996) and 
detailed elsewhere (Riederer et al. in preparation). Briefly, air was drawn using a GilAir® Plus low-flow 
pump (Sensidyne®, St. Petersburg, FL, USA) via polypropylene tubing into a 
MilliporeSigmaTMSupelcoTM CT420 proprietary glass thermal desorption tube optimized for terpene 
collection (MilliporeSigma, Burlington, MA, USA).  The tube was mounted on a metal stand, near the 
participant, at breathing height (~ 0.5 m above the ground). One field blank, consisting of a tube taken to 
the site and end caps momentarily opened but air drawn through was collected for each day of sampling, 
while field duplicates were collected on alternating weeks. Primary samples, blanks and duplicates were 

analyzed for 22 terpenes using thermal desorption-gas chromatography–mass spectrometry (TD-GC/MS) 
at Eurofins Laboratory (Edmonton, Alberta, Canada).  
Participants met research staff at a study site in Tacoma for informed consent, initial surveys, and 
measurements. Participants were then transported to Pack Forest (~one hr drive) in a vehicle fitted with a 
fan that blew filtered air into their seating area to reduce terpene exposure prior to their session. Upon 
arrival, they were outfitted with a PAPR with charcoal filter (‘zero filter’) to prevent terpenes inhalation 
during initial study protocols.  
Participants were then outfitted with the physiology equipment in a clinic room, rested for 10 
min, and completed a survey with affective assessments and the baseline blood draw before being 
transported to the forest site where the zero filter was changed to the randomly assigned filter. Blood 
pressure, HR, and self-reported affect and stress were assessed at the beginning (T1; 0-5 min) and end of 
the session (T4; 55-60 min). Self-reported affect and stress were also assessed at two additional points: T2 
(17.5-22.5 min) and T3 (37.5-42.5 min). A second blood sample was collected at T4. HF HRV and SCL 
were assessed continuously. A full daily protocol is outlined in Figure 1. 
 
Figure 1. Overview of procedure sequence for a study session. 

 
4. Relevant Scientific Background  
Nature contact has been linked to psychological and physiological well-being in human beings (Bratman 
et al., 2019). As outlined by Frumkin et al. (2017), these benefits include stress reduction (assessed via 
decreased heart rate, blood pressure, cortisol levels, and self-report measures), decreased anxiety, 
negative affect, and depressive symptoms, and increased positive affect, prosocial behavior, and 
attentional capacity (Beil & Hanes, 2013; Berman et al., 2008; Bratman et al., 2015; Duncan et al., 2014; 
Ewert & Chang, 2018; Hong et al., 2018; Hunter et al., 2019; Jiang et al., 2014; Kaźmierczak, 2013; 
McAllister et al., 2017; Piff et al., 2015; Putra et al., 2020; Ward Thompson et al., 2016; Zhao et al., 
2022). Additionally, nature contact has been associated with improved sleep, reduced mortality, and 
reduced risk of Type II diabetes (Astell-Burt et al., 2014; Astell-Burt & Feng, 2020; Bodicoat et al., 2014; 
Gascon et al., 2016; James et al., 2016; Johnson et al., 2018; Shin et al., 2020). Despite these 
demonstrated associations, the causal mechanisms that may be responsible for these effects are less well 
known. One possible pathway is reduction of stress and associated anti-inflammatory 
psychoneuroimmunological processes (Hartig et al., 2014; Kuo, 2015; Ulrich et al., 1991). 
Shinrin-yoku, also known as “forest bathing”, is a specific set of human-nature interactions that 
have been shown to improve mood, reduce anxiety and depressive symptoms, and have beneficial effects 
on HRV, blood pressure, HR, stress hormone levels, and inflammatory biomarkers (De Brito et al., 2020; 
Furuyashiki et al., 2019; Horiuchi et al., 2014; D.-S. Kim et al., 2015, 2015; Kobayashi et al., 2017, 2018; 
Lanki et al., 2017; J. Lee et al., 2015; Li et al., 2007, 2008; Mao, Cao, et al., 2012; Mao, Lan, et al., 2012; 
Ochiai et al., 2015; Oomen-Welke et al., 2022; B. J. Park et al., 2009; B.-J. Park et al., 2007; Song et al., 
2015, 2017; Stigsdotter et al., 2017; Tsunetsugu et al., 2013; Yu et al., 2017). However, some studies 
have not observed significant changes or reported contrary observations when comparing outcomes 
following forest exposure to earlier baseline measurements or outcomes following control exposure, 
including no significant difference or a difference against the hypothesized direction in mood, stress 
hormone levels, blood pressure, and HRV (De Brito et al., 2020; Furuyashiki et al., 2019; Gidlow et al., 

2016, 2016; Horiuchi et al., 2014; Kavanaugh et al., 2022; Y. Kim et al., 2022; Lanki et al., 2017; Mao, 
Cao, et al., 2012; Oomen-Welke et al., 2022; Stigsdotter et al., 2017; Yu et al., 2017).  
One possible pathway for the psychophysiological benefits of forest bathing is through exposures 
to terpene compounds that contribute to the smell of the forest (Li et al., 2008). Terpenes are types of 
biogenic volatile organic compounds (BVOCs). Over 55,000 different chemically structured terpenes 
have been identified, all generally with a (C5H8)n chemical formula, and classified by differences in linked 
isoprene units (Kanwal et al., 2022). Emitted by plants, terpenes aid in plant reproduction, defense, inter- 
and intraspecific interactions, and abiotic stress response (Dudareva et al., 2006).  
In animal and in vitro experiments, terpenes have demonstrated anti-inflammatory, anti-
tumorigenic, and neuroprotective action (Cho et al., 2017). As summarized by De Cássia Da Silveira E Sá 
et al. (2013) and Eddin et al. (2021), rats, mice, and guinea-pigs exposed to terpenes displayed anxiolytic 
and anti-inflammatory responses via reduced plasma corticosterone levels, reduced inflammation in  
colonic tissue, and a reduction in bronchial resistance, paw edema, and leukocyte, neutrophil, IL-1β, 
TNF-α, and IL-6 levels (Bastos et al., 2011; De Almeida et al., 2017; Nascimento et al., 2009; 
Saiyudthong & Marsden, 2011; Santos, 2004; Zhang et al., 2018). Additionally, terpene exposure was 
shown to increase white blood cell count, IL-2, IL-10, T cell, and lymphocyte levels, and antibody cell 
production in rats and mice (Badr et al., 2011; Raphael & Kuttan, 2003; Trinh et al., 2011).  
In human monocytes, terpenes can inhibit production of inflammatory cytokines, leukotrienes, 
and associated metabolites, including TNF-α, IL-1β, Il-4, Il-5, IL-6, IL-8, and leukotriene B4 (LTB4; De 
Cássia Da Silveira E Sá et al., 2013; Hart et al., 2000; Juergens et al., 1998, 2004). Additionally, terpene 
or essential oil exposure has been associated with anti-inflammatory effects in asthma patients, reduced 
anxiety and depressive symptoms, reduced stress, increased positive affect, and improved cognitive 
function and sleep (Chen et al., 2022; Fung et al., 2021; Goes et al., 2012; Juergens et al., 2003; Kerr et 
al., 2021; Koyama & Heinbockel, 2020; M. Lee et al., 2017; Moss & Oliver, 2012; Woo et al., 2023). 
These anti-inflammatory, anti-cancer, antioxidative, and neuroprotective properties of forest-derived 
terpenes may be mediated by signal transduction (i.e., a relay of signals within a cell) and changes in the 

production of transcription factor proteins that regulate gene transcription (i.e., NF-κB; Cui et al., 2022; 
D.-S. Kim et al., 2015; T. Kim et al., 2020; Latchman, 1993; Nair et al., 2019; Rufino et al., 2014).  
Aoshima and Hamamoto (1999) proposed several pathways through which fragrant compounds 
might affect the brain: stimulation of the olfactory system and resulting psychological effects, absorption 
into the blood via the lungs by respiration, skin application, and gastrointestinal exposure via food. 
Similar pathways for essential oil inhalation were summarized by Fung et al. (2021). With respect to the 
olfactory system: essential oil molecules can bind to chemoreceptors in the nasal cavity and activate 
neural signaling, resulting in psychological changes (Cui et al., 2022; Faturi et al., 2010; Kagawa et al., 
2003). Additionally, essential oil molecules may move through the olfactory system neuronal network via 
extracellular delivery or mucosa, arriving in the brain in a similar way to nasally delivered medications 
(Chioca et al., 2013; Cui et al., 2022; Fung et al., 2021; Hanson & Frey, 2008). From there, they may 
interact with transient receptor potential channels (TRP), gamma-aminobutyric acid, serotonin, and 
dopamine receptors (Hanson & Frey, 2008). Finally, essential oil molecules may also be transported via 
lung alveoli and blood to interact directly with the CNS (Chioca et al., 2013; Faturi et al., 2010; Fung et 
al., 2021; Kagawa et al., 2003). 
In this study, participants were exposed to terpenes via inhalation of ambient forest air. Most 
inhaled air enters through the nose, and although human beings switch to oral breathing during exercise, 
approximately 30-35% of air is still inhaled via the nose in those cases as well (Koenig, 2000). Sense of 
smell, or olfaction, refers to the sensory process through which the olfactory system and brain interpret 
odors from the surrounding environment (Doty, 2001). The olfactory epithelium that lines the interior of 
the nose consists of olfactory receptor neurons (ORNs) that bind with odorants (Purves & Williams, 
2001). ORN axons project sensory information to neurons in the olfactory bulb, which then project to the 
pyriform cortex in the temporal lobe, as well as the hypothalamus and the amygdala.  
Olfaction and affective responses are linked in experimental studies, in part because odors can 
directly elicit individual affective responses following exposure (Kontaris et al., 2020; Retiveau et al., 
2004; Seubert et al., 2008). Some odors may impair working memory, while others reduce anger and 

depressive symptoms, and improve mood (Komori et al., 1995; Martin & Chaudry, 2014; Retiveau et al., 
2004; Schiffman et al., 1995). As outlined by Durrant et al. (2016), the olfactory bulb might also be the 
site of an early innate immune response, as experimental studies of mice found olfactory bulb expression 
of proinflammatory cytokines, including TNF-α, TNFR1, IL-1β, IL-6, and IκB kinase (Aniszewska et al., 
2015; Leyva-Grado et al., 2009; Mori et al., 2005). In-vitro experiments have also shown that olfactory 
ensheathing cells (OECs) may activate NF-κB transcription factors, express Toll-like receptors (TLR) 2 
and 4, and express inducible nitric oxide synthase (iNOS) mRNA and enzymes in response to bacteria 
and pathogen incubation (Harris et al., 2009; Vincent et al., 2007).  
Here, we aim to fill a critical gap in knowledge by examining whether terpenes play a role in the 
health effects of nature contact in a real-world forest environment by assessing whether terpene 
inhalation, with a focus on the olfactory pathway, impacts stress reduction and affective outcomes using a 
randomized double-blind crossover trial design. The primary outcome was the HF component of HRV, a 
measure of parasympathetic nervous system (PNS) activity, or relaxation (add some citations to back up 
this claim). Secondary outcomes included measures of skin conductance levels (SCL), blood pressure, 
and HR (i.e., measures of sympathetic nervous system activity, or stress), self-reported stress and affect, 
and levels of inflammatory cytokines in serum. Terpene concentrations in serum were measured to assess 
the degree of association of absorbed dose with study outcomes. We predicted that exposure to forest 
terpenes would be associated with greater increases in HF HRV and positive affect, and greater decreases 
in SCL, blood pressure, HR, self-reported stress, negative affect, and levels of inflammatory cytokines in 
serum compared to the condition with no forest terpenes exposure. Additionally, we predicted that study 
outcomes would be associated with absorbed dose of forest terpenes.  
 
To our knowledge, no study has been conducted that concurrently investigated real-world 
ambient terpene exposure in a forest, absorption of these terpenes, and impacts of experimental vs. control 
conditions of these terpene exposures on psychophysiological and immunological effects in human 
beings. Therefore, this study aims to further scientific understanding of the role that forest terpene 
inhalation exposures play in the multisensory pathways that link human well-being and forest exposure. 

 
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