Endocannabinoid and Psychological Responses to Yoga in Healthy Adults
Phase: NA
Conditions: Healthy
Sponsor: University of Wisconsin, Madison

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Document Date: 3-06-2018 
 
PROTOCOL 
Project Description for IRB 
Title: Endocannabinoid and Psychological Responses to Yoga in Healthy Women 
 
 
Principal Investigator: 
Kelli F. Koltyn, Department of Kinesiology, UW-Madison 
 
 
Co-Investigators/Additional Study Team Members: 
Brianna Leitzelar, Department of Kinesiology, UW-Madison 
Kevin Crombie, Department of Kinesiology, UW Madison 
Cecilia Hillard, Department of Pharmacology, Medical College of Wisconsin 
 
 
Study Sites: 
University of Wisconsin-Madison (lead site) 
Medical College of Wisconsin (assays) 
 
 
Funding: This research will be supported by the UW-Madison Virginia Horne Henry Fund. 
 
 

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Study Objectives 
Yoga is an alternative form of exercise which has gained recent attention in the literature, 
ranging from children to older adults and healthy individuals to those with chronic illnesses. 
Physical benefits include improvements in anthropometric measures and blood pressure (Chu et 
al., 2016). Psychological benefits are demonstrated in a wide range of populations (e.g., Cramer 
et al., 2013; Hofmann et al., 2016), however these outcomes are less established. Further, there is 
relatively little known about the acute outcomes of participating in yoga, thus an understanding 
of the immediate impact of yoga is warranted. Further, it is important to understand the 
mechanisms behind these effects to develop protocols that are able to maximize the potential of 
yoga. One recent system proposed to be involved in the benefits of exercise is the activation of 
the endocannabinoid (eCB) system (Sparling et al., 2003). However, this has not been expressly 
tested using yoga. Thus, an exploratory pilot study is warranted to examine the acute responses 
of the eCB system to yoga as well as to understand the acute impact of yoga on mood. 
 
Primary Aim: To determine the magnitude of change of eCBs (AEA, 2-AG) pre- to post- 
yoga and pre- to post- quiet rest (QR).  
 
Secondary Aim: To determine the magnitude of change in mood (POMS- total mood 
disturbance; STAI- state anxiety; SF-MPQ- overall pain) pre- to post- yoga and pre- to 
post- QR.    
 
Study Significance 
In the last 15 years, research determining the impact of yoga on physical and mental health has 
grown exponentially (Cramer et al., 2014). However, much of this research examines the 
benefits of long-term training, thus the acute effects of yoga are relatively unknown. Physical 
benefits of yoga training interventions include decreased body mass index (BMI), improved 
blood pressure, cholesterol, body weight, and triglycerides, as well as decreased heart rate (Chu 
et al., 2016). In addition, improvements in mental health such as anxiety in a healthy population 
(Hofmann et al., 2016), depression in individuals diagnosed with depression (Cramer et al., 
2013), fatigue in women with breast cancer (Sadja et al., 2013), and pain in adults with low back 
pain (Goode et al., 2016) have been demonstrated. However, many of these studies report a need 
for further evidence before strong conclusions can be drawn.  
 
Surprisingly few studies have examined the acute effects of yoga, thus the immediate experience 
of individuals practicing yoga are not well understood. Two recent studies demonstrated the 
acute benefits of yoga including decreased negative affect,  increased positive affect (Gaskins et 
al., 2014; Szabo et al., 2017), and decreased state anxiety (Szabo et al., 2017). However, 
Gaskings and colleagues (2014) examined both acute and chronic effects of yoga and found no 
changes in perceived stress for either. Although these studies provide some insight, future 
research is required to replicate and expand this knowledge base.  
 
There is even less known about the mechanisms behind the effects of participating in yoga. 
However, since yoga can be considered a type of exercise, we can draw from the mechanistic 
work in the exercise realm. One of the most recently proposed mechanisms is that of the 
endocannabinoid (eCB) system due to its role in several psychological constructs, such as 
depression and anxiety (Hill et al., 2009; Hill et al., 2008; Hillard et al., 2014; Mechoulam et al., 

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2013; Riebe et al., 2011). The eCB system is a neuromodulatory system involved in numerous 
processes including inflammation, neurogenerative diseases, cognition, reward, memory, the 
stress response, and psychological disorders (Hillard, 2015; Mechoulam et al., 2013; Reibe et al., 
2011). In brief, the eCB system consists of the cannabinoid receptors CB1 and CB2 as well, their 
effectors, N-arachidonylthanolamine (AEA, “anandamide”) and 2-arachidonolyglycerol (2-AG), 
and the enzymes that degrade them (fatty acid amide hydrolase, FAAH; monoacylglycerol 
lipase, MAGL).  
 
There is evidence that exercise impacts endogenous eCB signaling throughout the body 
(Tantimonaco et al., 2014). Sparling and associates (2003) were the first researchers to report 
increases in eCBs following 50 minutes of moderate cycling or running. Since then, strenuous 
hiking (Feurecker et al., 2012), intense cycling (Heyman et al., 2012), and treadmill running 
(Raichlen et al., 2012; Raichlen et al., 2013) have also been shown to influence the 
endocannabinoids. Further, recent work in our lab has demonstrated increases in eCBs following 
isometric exercise (i.e., static muscle contractions), and this was in conjunction with reductions 
in pain (Koltyn et al., 2014). However, no study, to our knowledge, has examined the eCB 
system in response to yoga.  
 
Since it is currently unknown whether yoga influences the eCB system, an exploratory pilot 
study will be conducted to examine the eCB and psychological responses to yoga. Thus, we may 
determine the effect sizes for the proposed outcomes in order to sufficiently power subsequent 
studies. 
 
Research Design and Procedures 
The proposed study is an exploratory pilot study where participants will complete control and 
experimental conditions.  
 
Subject Population 
Twelve women from the University of Wisconsin-Madison will be recruited to participate in this 
study. Participants’ ages will range from 18-45 years and will report being healthy. 
 
Sample Size Justification 
Based on our previous research showing a large effect of exercise on eCBs, a statistical power 
analysis was performed for sample size estimation based on a large effect size. Using Cohen’s f 
effect size (ES) of .4, alpha = .05 and power = 0.80, the total sample size needed for this study is 
N = 10 (GPower 3.1) for a within subject repeated measures design. To account for participant 
attrition, up to 12 participants will be recruited.    
 
Inclusion Criteria 
Inclusion criteria will be as follows: 
 
• 
> 18 years old and < 45 years old and 
• 
report being healthy. 
 
 
 

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Exclusion Criteria 
Exclusion criteria will include: 
• 
Being pregnant or planning to become pregnant, 
• 
currently smoking, 
• 
having a history of light headedness or fainting during blood draws or physical 
activity, 
• 
having a history of chest pain during physical activity, 
• 
having a bone, joint, cardiac, or other medical condition that a doctor has said 
may be worsened by physical activity, 
• 
taking medications for any chronic diseases such as high blood pressure or 
diabetes, 
• 
responding ‘Yes’ to any of the seven questions on the Par-Q. 
 
Identification and Recruitment of Subjects 
Volunteers will be recruited from students, faculty, and staff at the University of Wisconsin-
Madison to participate in this study. They will be between the ages of 18 and 45 and report being 
healthy. Participants will be recruited through flyers and direct recruitment. Flyers will be posted 
in approved locations across the UW campus. In addition, a study team member will directly 
recruit participants from lecture halls by making an announcement and handing out flyers. The 
instructor’s consent will be sought before the team member attends the lecture. Volunteers will 
complete a phone screen prior to Study Visit #1 to determine eligibility. These recruitment 
methods were effective in previous studies conducted in the Exercise Psychology Laboratory. 
  
Instruments 
Demographic Form. The demographic form will consist of multiple choice and short answer 
questions (e.g., age, sex, ethnicity, etc.) and will be used to describe the sample. 
 
Heart Rate. Heart rate will be collected to quantify the intensity of the study tasks using a heart 
rate monitor. 
 
Profile of Mood States. The POMS (McNair, Lorr, & Droppleman, 1971) is a 65-item 
questionnaire using a 5-point Likert-type scale ranging from 0 “not at all” to 4 “extremely.” It 
will be used to determine the acute mood state of participants which include: tension, depression, 
confusion, anger, fatigue, vigor, and total mood disturbance. Internal consistencies for the 
subscales on the POMS range from .84 to .95 (McNair et al., 1971).  
 
Ratings of Perceived Exertion (RPE). A validated RPE scale (Borg et al., 1998) will be used to 
measure the perceived intensity of the exercise. The scale ranges from 6 “no exertion at all” to 20 
“maximal exertion.”  
 
Short-Form McGill Questionnaire. The SF-MPQ (Melzack, 1987) is a 15-item survey with 
items rated on a 4-point scale ranging from 0 “none” to 3 “severe.” Eleven items assess the 
intensity of sensory components of pain and four items assess the intensity of affective 
components of pain. In addition, a Present Pain Index (PPI) and a Visual Analogue Scale (VAS) 
are included in the questionnaire.  
 

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State-Trait Anxiety Inventory. The STAI Form Y-1 (Spielberger et al., 1983) is a 20-item 
questionnaire designed to measure state anxiety. Items ranged from 1 “almost never” to 4 
“almost always” with higher scores indicating higher anxiety. The internal consistency for the 
STAI ranges from .86 to .95 (Spielberger et al., 1983).  
 
Procedures 
Prior to attending the study visits, volunteer eligibility will be determined through a preliminary 
phone screen. If the volunteer is eligible, they will be scheduled for Study Visit #1 and will be 
asked to refrain from exercise for 24 hours and to refrain from drinking or eating (except for 
water)  for 2 hours prior to their study visit. Participants will attend a total of two study visits, 
each 60-75 minutes long, to complete both the control and experimental sessions. There will be 
between 2 and 7 days between Study Visit #1 and Study Visit #2. All visits will be completed 
independently and participants will spend a total of 120-150 minutes completing this study. 
Participants will be randomly assigned to the sessions.  
 
Experimental Task: The experimental task will consist of 40 minutes of yoga in a sound-
dampened chamber. Participants will follow an online yoga video through YouTube and 
will be instructed to participate to the best of her ability. Heart rate (HR) will be collected 
every five minutes and the session intensity will be recorded through an over-all rating of 
perceived exertion at the end of the session (RPE; Borg et al., 1998). A study team 
member will monitor the participant via a window into the sound-damped chamber.  
 
Control Task: The control task will consist of 40 minutes of quiet rest in a sound-
dampened chamber. Participants will not be allowed to use electronics during this 
session, however reading material will be made available. HR will be collected every five 
minutes and RPE will be recorded at the end of the session. A study team member will 
monitor the participant via a window into the sound-damped chamber. 
 
Visit 1 
Participants will be randomly assigned to completing the experimental or control task prior to 
coming in for Study Visit #1. All study visits will be completed in the Exercise Psychology 
Laboratory in the University of Wisconsin-Madison Natatorium building. Upon arrival, 
participants will enter a sound-damped chamber to ensure privacy. They will complete an 
informed consent form and a packet of questionnaires consisting of: the demographic 
questionnaire, Profile of Mood States (POMS; McNair et al., 1992), State-Trait Anxiety 
Inventory (STAI; Spielberger et al., 1983), and the Short-Form McGill Pain Questionnaire (SF-
MPQ; Melzack, 1987). Upon completion of questionnaires, participants will have their age, sex, 
weight, and height measurements assessed. Participants will then be informed of their task and 
will be outfitted with a heart rate monitor and a study team member will explain the RPE scale. 
Next, participants will have their blood drawn (5ml) immediately before completing the visit 1 
task (experimental or control). Immediately upon task completion, the participants will be asked 
for their RPE, have their blood drawn (5 ml) and will complete the same questionnaires listed 
previously (POMS, STAI, SF-MPQ, and VAS-F). Blood samples will be prepared for analysis 
within 10 minutes of collection (see ‘Endocannabinoid Assays’ section below).    
 
 

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Visit 2 
Participants will complete the task that was not assigned to them during study visit #1 (control or 
experimental task). Although they will not complete the informed consent or demographic 
questionnaire, all procedures will be the same. Participants will complete the following 
questionnaires pre- and post-task completion: POMS, STAI, and SF-MPQ and will be asked 
their RPE. Further, two separate blood samples (5ml) will be taken immediately pre- and post- 
task completion and will be processed within 10 minutes of collection. Participants will receive 
$30 gift card upon completion of both study visits. 
 
Endocannabinoid Assays 
Blood samples will be collected using EDTA containing tubes (BD Vacutainer, K3E EDTA K3) 
and centrifuged within 10 minutes of collection. Separated plasma will be frozen at -80 C until 
analysis. Analysis will be conducted at the Medical College of Wisconsin under the supervision 
of Dr. Cecilia Hillard. The analysis will quantify the plasma concentrations of eCBs (AEA and 
2-AG) and their related biolipids (PEA, OEA, 2-OG) using an isotope-dilution, electrospray 
ionization liquid chromatography/mass spectrometry of the daughter ions (LC-ESI-MS-MS). 
This protocol has been previously described (Koltyn et al., 2014).  
 
Data Analysis 
The design of this study is a within subject, repeated measure design where participants complete 
pre-post measures during two study visits. Since this is an exploratory pilot study, our aims are to 
determine the magnitude of change of our outcomes, using Cohen’s d.  Specifically, effect sizes 
for the change in eCBs (AEA and 2-AG) pre- to post- condition will be determined for yoga and 
QR. In order to meet the secondary aim, Cohen’s d will be conducted for each of the 
psychological outcomes (POMS-total mood disturbance; STAI-state anxiety; SFMPQ- pain) to 
determine the pre- to post-changes for each task (yoga, QR).  
 
Data Collection 
Data will be collected through self-report and biological samples. The only identifiable 
information will be collected through the informed consent document. The informed consent 
documents (hard copy), which include the participant’s assigned identifier, will be stored in a 
locked cabinet in the PI’s office located in the Kinesiology Department at the University of 
Wisconsin-Madison Natatorium building. These will be separate from the other study documents 
and only the PI will have access. All data will be coded using a personal identifier assigned to 
the participant after informed consent is signed, thus all collected data will be coded. All 
information collected on paper (i.e., age, HR, height, RPE, weight), besides the informed 
consent, will be stored in a locked cabinet in the Exercise Psychology Lab, which is part of the 
Kinesiology Department.  Self-report data (questionnaires) will be collected electronically 
through Qualtrix and only the research team will have access to both electronic and hard-copy 
data. Raw data will be input into a computer for data analysis where it will be stored in a secured 
file on the Exercise Psychology Lab server which is located in a password protected area with 
firewall protection and backed up daily. All team members will have access to the coded 
information if located in the Exercise Psychology Laboratory. Biological samples (blood 
samples) will be stored in a freezer in the Biodynamics Laboratory located in the Natatorium at 
the University of Wisconsin-Madison after which they will be transferred to a container of dry 
ice and driven by the PI to Dr. Hillard’s laboratory at the Medical College of Wisconsin. The 

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blood samples then will be stored in Dr. Hillard’s lab until eCB assays are performed. They will 
be exhausted during the eCB extraction processes. The blood samples then will be stored in Dr. 
Hillard’s lab after which they will be exhausted. The blood samples will contain only the 
participant ID number, time of collection, and session date; thus, participants will not be able to 
be identified. Moreover, the screening data for eligible participants will be kept and secured in a 
locked file cabinet in the Exercise Psychology Lab until the participant has either completed the 
study, removed themselves from participation, or is excluded from participation. All screening 
data for eligible participants will be shredded upon participant completion, dropout, or exclusion 
of the study. Phone screening data from any interested potential participants not meeting 
inclusion criteria will be shredded immediately. Biological samples will be exhausted upon 
completion of endocannabinoid assay. Raw data will be shredded after 7 years and coded data 
will be kept indefinitely.  
 
Sample/Specimen Collection 
Blood samples will be collected by a trained phlebotomist using EDTA containing tubes (BD 
Vacutainer, K3E EDTA K3) and centrifuged. Plasma will be frozen at -80 C in a freezer in a 
designated area on the UW-Madison campus until all samples are collected. All samples will be 
shipped to Dr. Hillard’s lab at the Medical College of Wisconsin and will be stored in a freezer 
until analysis. No samples will include identifiable information and the results from the eCB 
assays will be shared between study team members using the participant ID numbers 
 
 
 
 
 
 

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References 
Borg, G. (1998). Borg's perceived exertion and pain scales. Human kinetics. 
Chu, P., Gotink, R. A., Yeh, G. Y., Goldie, S. J., & Hunink, M. M. (2016). The effectiveness of 
yoga in modifying risk factors for cardiovascular disease and metabolic syndrome: a 
systematic review and meta-analysis of randomized controlled trials. European journal of 
preventive cardiology, 23(3), 291-307. 
Cramer, H., Lauche, R., & Dobos, G. (2014). Characteristics of randomized controlled trials of 
yoga: a bibliometric analysis. BMC complementary and alternative medicine, 14(1), 328. 
Feuerecker, M., Hauer, D., Toth, R., Demetz, F., Hölzl, J., Thiel, M., ... & Chouker, A. (2012). 
Effects of exercise stress on the endocannabinoid system in humans under field 
conditions. European journal of applied physiology, 112(7), 2777-2781. 
Gaskins, R., Jennings, E., Thind, H., Becker, B., & Bock, B. (2014). Acute and cumulative 
effects of vinyasa yoga on affect and stress among college students participating in an 
eight-week yoga program: a pilot study. International journal of yoga therapy, 24(1), 63-
70. 
Goode, A. P., Coeytaux, R. R., McDuffie, J., Duan-Porter, W., Sharma, P., Mennella, H., ... & 
Williams, J. W. (2016). An evidence map of yoga for low back pain. Complementary 
therapies in medicine, 25, 170-177. 
Heyman, E., Gamelin, F. X., Goekint, M., Piscitelli, F., Roelands, B., Leclair, E., ... & Meeusen, 
R. (2012). Intense exercise increases circulating endocannabinoid and BDNF levels in 
humans—possible implications for reward and depression. Psychoneuroendocrinology, 
37(6), 844-851. 

9 
 
Document Date: 3-06-2018 
 
Hill, M. N., Miller, G. E., Carrier, E. J., Gorzalka, B. B., & Hillard, C. J. (2009). Circulating 
endocannabinoids and N-acyl ethanolamines are differentially regulated in major 
depression and following exposure to social stress. Psychoneuroendocrinology, 34(8), 
1257-1262. 
Hill, M. N., Miller, G. E., Ho, W. S., Gorzalka, B. B., & Hillard, C. J. (2008). Serum 
endocannabinoid content is altered in females with depressive disorders: a preliminary 
report. Pharmacopsychiatry, 41(02), 48-53. 
Hillard, C., & Liu, Q. S. (2014). Endocannabinoid signaling in the etiology and treatment of 
major depressive illness. Current pharmaceutical design, 20(23), 3795-3811. 
Hofmann, S. G., Andreoli, G., Carpenter, J. K., & Curtiss, J. (2016). Effect of Hatha yoga on 
anxiety: a meta‐analysis. Journal of Evidence‐Based Medicine, 9(3), 116-124. 
Koltyn, K. F., Brellenthin, A. G., Cook, D. B., Sehgal, N., & Hillard, C. (2014). Mechanisms of 
exercise-induced hypoalgesia. The Journal of Pain, 15(12), 1294-1304. 
McNair, D., Lorr, M., & Droppleman, L. (1971). Mannual for the profile of mood states 
(POMS). San Diego, CA: Educational and Industrial Testing Service. 
Mechoulam, R., & Parker, L. A. (2013). The endocannabinoid system and the brain. Annual 
review of psychology, 64, 21-47. 
Melzack, R. (1987). The short-form McGill pain questionnaire. Pain, 30(2), 191-197. 
Raichlen, D. A., Foster, A. D., Gerdeman, G. L., Seillier, A., & Giuffrida, A. (2012). Wired to 
run: exercise-induced endocannabinoid signaling in humans and cursorial mammals with 
implications for the ‘runner’s high’. Journal of Experimental Biology, 215(8), 1331-1336. 

10 
 
Document Date: 3-06-2018 
 
Raichlen, D. A., Foster, A. D., Seillier, A., Giuffrida, A., & Gerdeman, G. L. (2013). Exercise-
induced endocannabinoid signaling is modulated by intensity. European journal of 
applied physiology, 113(4), 869-875. 
Riebe, C. J., & Wotjak, C. T. (2011). Endocannabinoids and stress. Stress, 14(4), 384-397. 
Sadja, J., & Mills, P. J. (2013). Effects of yoga interventions on fatigue in cancer patients and 
survivors: a systematic review of randomized controlled trials. Explore: The Journal of 
Science and Healing, 9(4), 232-243. 
Sparling, P. B., Giuffrida, A., Piomelli, D., Rosskopf, L., & Dietrich, A. (2003). Exercise 
activates the endocannabinoid system. Neuroreport, 14(17), 2209-2211. 
Spielberger, C. D., Gorsuch, R. L., Lushene, R., Vagg, P. R., & Jacobs, G. A. (1983). Manual for 
the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting Psychologists Press.Szabo, 
A., Nikházy, L., Tihanyi, B., & Boros, S. (2017). An in-situ investigation of the acute 
effects of Bikram yoga on positive-and negative affect, and state-anxiety in context of 
perceived stress. Journal of Mental Health, 26(2), 156-160. 
Tantimonaco, M., Ceci, R., Sabatini, S., Catani, M. V., Rossi, A., Gasperi, V., & Maccarrone, M. 
(2014). Physical activity and the endocannabinoid system: an overview. Cellular and 
molecular life sciences, 71(14), 2681-2698.