Can You Breathe Your Way To Better Health?

The purpose of this clinical trial is to explore the effects of either a 2-week high-intensity interval training (HIIT) or breath training intervention on measures of overall health, circulating biomarkers of stress, and immune function.

Specific aims include: - Does a 2-week HIIT or breath training intervention improve measurements of overall health, including heart rate variability, physical activity, sleep quality, and severity of depression, anxiety, and stress? -Does a 2-week HIIT or breath training intervention improve circulating concentrations of stress-related biomarkers? Does a 2-week HIIT or breath training intervention improve immune function? Researchers will compare HIIT and breath training to see if equivalent immune improvements are observed.

Participants will: -Undergo 2 weeks of HIIT or breath training interventions at a frequency of 3 times per week for 30 minutes or 5 times per week for 5 minutes if placed into an intervention group. -Undergo testing measures at the two pre- and post-intervention time points, if placed in the intervention groups or the healthy control group.

Study Overview

• Status: Enrolling by invitation
• Conditions: Cancer; Autonomic Dysfunction; Chronic Stress
• Intervention / Treatment: Behavioral: Hight intensity interval training; Behavioral: Cyclic Hyperventilation with Retention

Detailed Description

STUDY DESIGN:

This study uses a quasi-randomized control trial design, where N=36 will be randomly assigned into one of three groups. These groups will consist of the HIG (n =12), CHG (n= 12), and COG (n = 12). Each participant will receive an identification number as they enroll into the study and then will be quasi-randomly assigned in a balanced manner (with equal males and females in each group) to one of the three groups by a researcher not directly associated with the study. This person will use the excel randomization procedure. An overview of the study is presented in Figure 1 below. Participants will be randomly assigned into one of the three intervention groups: 1) high intensity group (HIG, n = 12), 2) breathe retention group (CHG, n = 12), 3) control group (COG, n = 12).

Figure 1. Overview of Study Visits

Figure 1: Study Overview This study will consist of three intervention groups denoted as HIG (HIIT group), BRG (breath retention group), and COG (control group). Upon completion of the pre-intervention data collection visits, participants will be placed into one of the three intervention groups using a quasi-randomized order. Researchers overseeing the study ensured all three intervention groups were matched to the best of their ability. A total of four testing visits, 2 pre-intervention and 2 post-interventions. The color coding used for the outline visits above demonstrates the similarities between data collection visits. Similar data collection methods for Visit 1 and Visit 3 will be used, as well as Visit 2 and Visit 4, as outlined above.

PARTICIPANTS:

Healthy participants consisting of males and females (N=60, HIG=20, BRG= 20, CON=20) over the age of 18 years are eligible for this study. Participants will be recruited using flyers and word of mouth at The University of Northern Colorado. The participants' age range will be from 18 years old to 50 years old. Participants will be required to complete the physical activity readiness questionnaire (PAR-Q+) to ensure safety while exercising. Inclusion and exclusion criteria are presented in Table 1. To assess the effectiveness of the intervention to potentially reduce stress, participants will be excluded based on their depression, anxiety, and stress scale (DASS-21) subcategory scores and must be experiencing mild to extremely severe depression, anxiety, and stress3. Participants must score a 10 or higher for depression, an 8 or higher for anxiety, and a 14 or higher for stress on the DASS-21.

Table 1: Inclusion and exclusion criteria Inclusion Criteria Age 18-50 years Sex Male and Female Informed Consent, the capability and willingness to give written informed consent, to understand the exclusion criteria, and to accept the randomized group assignment is required.

PAR-Q+, the capability and willingness to complete the physical activity readiness questionnaire, with no medical clearance needed.

Physical Activity, participants must refrain from regular HIIT and structured breathing practices for a month prior to participation Depression Anxiety and Stress Scale Subcategory Scoring, participants must score in the mild or greater category for Depression, Anxiety, and Stress. Depression ≥ 10, Anxiety ≥ 8, and Stress ≥ 14.

Exclusion Criteria:

Age Individuals under 18 years and over 50 years Significant Respiratory Conditions including but not limited to asthma, chronic obstructive pulmonary disease, Significant Cardiovascular Disease, participants with a known cardiovascular condition such as a previous myocardial infarction, congestive heart failure, stroke or transient ischemia attack, cardiomyopathy, serious arrythmias, peripheral vascular disease, untreated atherosclerosis, or hypertension Significant Musculoskeletal disease, including but not limited to osteopenia, osteoporosis, rheumatoid arthritis, and sarcopenia Cognitive and Mental Health, including but not limited to untreated anxiety, untreated depression, post-traumatic stress disorder, thoughts of suicide.

VISIT 1/VISIT 3 Surveys Upon arrival at Gunter Hall 1610, subjects will be given time to review the consent form on paper that will be supplied at this time. The investigator will explain the experimental protocol and invite the participant to ask questions. If the subject agrees to participate in the study, the subject and the researcher will sign two copies of the informed consent. One copy will be supplied to the participant, the second copy will remain in the investigators records. Subjects will also be notified that their full name and address will be collected to receive compensation at the end of this study per university policy.

Participants will then be asked to fill out surveys to assess participant health prior to starting the intervention. Surveys include PAR-Q+, Dass-21, Pittsburg Sleep Quality Index (PSQI), and Wisconsin Upper Respiratory Symptom Survey (WURSS). A description of the surveys is provided below. These surveys will be provided to each participant in both digital and physical form and participants will complete all survey questions. Following the completion of the surveys, the research team will collect a blood sample from the participant via a venous puncture by a trained phlebotomist for immune function analysis and hormone composition. An online version of the surveys will be generated using Qualtrics (Qualtrics.com, Seattle, WA).

Physical Activity Readiness Questionnaire for Everyone (PAR-Q+): PAR-Q+ utilizes yes or no generalized health questions to screen participants interested in engaging in an exercise program for the need of medical clearance and physician release. The PAR-Q+ has 7 general health questions ranging from "Do you feel pain in your chest at rest, during your daily activities or living, OR when you do physical activity?" to "Has your doctor ever said that you should only do medically supervised physical activity?"4. Individuals who are eligible for this investigation require no further medical clearance prior to participating in an exercise program and should truthfully answer NO to every question4.

Depression Anxiety and Stress Scale (DASS-21): The DASS-21 is made up of 21 questions and explores the participants depression, anxiety, and stress3,5. The questionnaire will request the participant reflect on the past week and each question will be answered on a 0-3 scale, with 0 meaning "Did not apply to me at all" and 3 meaning "Applied to me very much most of the time". Results of the DASS-21 will break down into three subcategories for the mental health parameters with 7 questions contributing specifically to each of the categories. Examples of questions covered in the DASS-21 include "I felt down-hearted or blue", "I felt I was close to panic", and "I felt I wasn't worth much as a person"3,5. Subcategories will then score to determine the severity of depression, anxiety, and stress on a five-point scale from "Normal" to "Extremely Severe"3,5.

Pittsburg Sleep Quality Index (PSQI): The PSQI consists of 19 questions which address the participants subjective sleep quality, sleep latency, sleep duration, efficiency of habitual sleep, disturbance of sleep, the use of sleeping medications, and daytime dysfunction6.

Wisconsin Upper Respiratory Symptom Survey (WURSS-21): The WURSS is an illness-specific quality of life measurement, with the purpose of assessing the impact of the common cold (acute upper respiratory infection). The WURSS-21 is composed of 21 questions investigating the perception of sickness, severity of sickness related symptoms over the last 24 hours, the extent that sickness has hindered the ability to think clearly and accomplish daily tasks over the past 24 hours, and the comparison of sickness to the day prior7.

Dietary Assessment: Participants' dietary habits will be assessed with a 24-hour dietary recall. Each participant will record what they ate and drank for 24 hours prior to Visit 1 and will be instructed to eat and drink the same foods and drinks at the same times prior to Visit 4. The 24-hour dietary recall will be turned into the research team at Visit 1 and Visit 4.

¬Anthropomorphic Assessments Bodyweight and Height Assessment: Participants will be instructed to remove their shoes, socks, and additional clothing other than their base layer prior to the weight and height assessment. Both measurements will be obtained via a stadiometer SECA 220 (Chino, California, USA) and the Detecto standing digital scale (Webb City, Missouri, USA). This measure will be used for descriptive purposes and will only be complete in Visit 1.

Air Displacement Plethysmography: Body composition will be assessed via air displacement plethysmography through utilization of the BODPOD (COSMED USA Inc., Concord, CA). Measurements obtained include body composition, lean body mass (LBM) and percent body fat (BF%). Participants will be instructed to remove their shoes, socks, jewelry, and any additional clothing other than their base layer. Participants will be instructed to wear a swim cap and body composition analysis will be performed per the manufacturer's guidelines8. This measure will be used for descriptive purposes and will only be complete in Visit 1.

Physiological Assessments Hydration Assessment: After completing the online inform consent and surveys, for Visit 1 and at the beginning of Visit 2, Visit 3, and Visit 4 urinalysis will be completed. Participants will be instructed to void their bladder into a specimen collection container. The urine sample provided will be analyzed for urine specific gravity using the PAL-10S (4410) urine specific gravity refractometer (ATAGO, Tokyo, Japan). A hydration cut off with be set at urine specific gravity (USG) <1.020. Participants arriving in a dehydrated state (USG>1.020) will be instructed to hydrate, if that is not possible the participant will be rescheduled and instructed to return at a later date.

Physical Activity and Sleep Tracking: The Whoop Strap 4.09. will be used to collect these data. Measures obtained from the Whoop strap include continuous heart rate monitoring, HRV, sleep cycle, sleep stage, and physical activity data. The HRV measurement from the whoop strap will then be compared to the HRV analysis performed using the standard deviation of normal-to-normal beat method10. This strap will be worn for the entire 2 week intervention period.

Immunological Assessments Blood Sampling: All blood collection procedures performed in this study will be performed by a trained phlebotomist in a controlled environment. Blood draws will occur at Visit 1 and Visit 3, at the pre intervention and post intervention timepoint respectively. All samples will be collected with the participant in a fasted state between the hours of 0600 - 1000. Participants will be asked to disclose their previous days dietary intake and will be asked to consume the same diet for 24 hours prior to their post intervention blood sampling. With each blood draw, the participant will donate approximately 40ml of blood via intravenous blood sample (approximately 2.5 tablespoons of blood). Blood will be collected from an arm vein using Safety-Lok butterfly needles collected into vacutainer collection vials. Blood samples will be evaluated for biomarkers of inflammation, brain health and stress as well as used to assess immune function.

VISIT 2/VISIT 4:

Hydration Assessment: As outlined above. Heart Rate Variability: HRV will be measured both using the WHOOP Strap (Whoop-inc., Boston, MA) and validated by manual assessment9-11. Participants will be attached to telemetry and the Standard deviation of normal-to-normal heartbeat method for HRV assessment will be used. The HRV measurement will be performed by attaching the participant to a 12 lead electrocardiogram (ECG) and a 5-minute recording will take place.11 Electrodes will be placed in accordance to the normal 12 lead ECG, including the following leads: right arm (RA), left arm (LA), right leg (RL), left leg (LL), V1, V2, V3, V4, V5, and V6. Lead II will be used and standard deviation of normal heartbeat to normal heartbeat will be assessed and will be reported in milliseconds (ms). Participants will then be categorized by their HRV range: HRV under 50ms are considered unhealthy, 50-100ms have compromised health, and above 100ms is considered healthy12. HRV will be assessed as a clinical indicator of systemic stress state and will be measured at the pre intervention and post intervention time point respectively. The total time for participant preparation and ECG recording will take less than 10 minutes.

VO¬¬2Max¬: This measure will be assessed on a cycle ergometer (Monark Ergomedic 895E, Monark, Varberg, Sweden) using a TrueOne 2400 Metabolic Measurement System (MMS-2400) (Parvomedics, Sandy, UT, USA)13. Participants will be outfitted with a Polar Heart Rate Sensor H1 (Polar Electro Inc., Bethpage, NY, USA) and instructed to rest in a seated position quietly with legs uncrossed for 5 minutes13. Following the 5 minute rest, baseline resting heart rate (RHR), resting blood lactate (RBL) via the Lactate Plus (NOVA Biomedical, USA) and resting blood pressure (RBP) using a Littman Stethoscope (3 M Centennial, CO, USA) and sphygmomanometer (American Diagnostic Corp., Hauppauge, NY, USA) will be obtained13. After vitals are obtained, the cycle ergometer will be fitted to the participants specifications and the participant will perform a brief warmup at a resistance of 0.5kp, a cadence of 60rpm for 5 minutes13. Immediately after the warm-up, participants will be instructed to increase their pedal cadence to 75-85rpm to begin stage 113. At the start of every 3-minute stage, 0.5kp of resistance will be then added to cycle ergometer weight basket13. At the end of every stage, heart rate (HR), blood pressure (BP), blood lactate (BL), and rate of perceived exertion (RPE) will be measured13. Once BL reached the onset of lactate accumulation (OBLA; >4mmol), incremental workload will increase by 0.2kp to ensure maximal ¬VO2max13. Termination of the test will occur in response to volitational fatigue or a decrease of pedal cadence below 75rpm14. This is a descriptive measure and will only be determined in Visit 2.

INTERVENTION INSIGHT:

Exercise Intervention: During the exercise session HR will be measured via Polar heart rate strap (Kempele, Finland). The training sessions will consist of six, 90-second high intensity cycling (HIC) sprints performed on a cycle ergometer (Monark Ergomedic 895E, Monark, Varberg, Sweden) at 80-90% VO2max, followed by 180 seconds of low intensity cycling (LIC) at 50-60% of VO2max. During both HIC and LIC intensities, participants will be asked RPE, which will be compared to the participants heart rate throughout the exercise sessions. This supervised training protocol will require 9 minutes of HIC at 80-90% VO2max and 18 minutes of LIC at 50-60% VO2max. The cycling session will begin with a brief warm up and end with a cool down totaling the entire supervised exercise session for 30 minutes. Training sessions will be performed 48 hours post the previous training session for a total of 3 times per week on Mondays, Wednesdays, and Fridays. Immediately after completion of the first and sixth training session HR recovery (HRr) will be assessed for standardized duration of 5 minutes.

Breathing Intervention: The breathing intervention will consist of a once daily breathing practice lasting about 5 to 10 minutes for 5 days per week. Breathing practice is a form of cyclic hyperventilation consisting of 30 breath (inhalation and exhalation) repetitions followed by an exhaled breath retention for 15 seconds during the first week and up to 30 seconds during the second week. A total of 3 rounds will be performed by the participants. Participants will be instructed to perform breathing repetitions in a controlled and consistent manner while either seated or lying down. Participants will receive guided instruction via a video on breath cycle queues, informing the participants when to inhale, exhale and when to retain their breath while being supervised by the research team via zoom call9. Upon completion of the breath training sessions the research team will conduct a guided cooldown and check in with all participants to ensure they are feeling no adverse side effects from the breathing exercise. The guided cooldown will consist of a member of the research team instructing participants to gradually return to their normal respiration rate upon completion of the breathing protocol through ques such as now we are going to take some relaxing deep breathes, full inhalation through the nose, followed by full exhalation through the mouth, while gradually increasing the duration of both inhale and exhale. The occurrence of adverse symptoms or events associated with the intervention will be recorded. All participants will be reminded that their involvement in the training intervention is voluntary, and they can withdraw at any time. Immediately after completion of the first and tenth CHR session HRr will be assessed for a standardized duration of 5 minutes.

Control Group: The control group will undergo the same testing procedures outlined above, but they will be asked to maintain their current level of activity and normal daily habits for the duration of the intervention.

ANALYSIS:

All data will be collected and presented as group means and standard deviation. Statistical analysis will be performed to determine whether there are significant differences between intervention groups at the pre-intervention time point. All data will be evaluated for independence of observations, normal distribution of samples, and variance amongst samples. Then, a 3(group) x 2 (timepoint) repeated measures ANOVA will be used to assess statistical significance between and within participants of each group. Significance will be set at p<0.05.

• Study Type: Interventional
• Enrollment (Estimated): 36
• Phase: Not Applicable

Contacts and Locations

Study Locations

• United States
  • Colorado
    • Greeley, Colorado, United States, 80631
      • University of Northern Colorado, 1610 Gunter Hall, 1828 10th Ave, Greeley, CO 80631

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Age, 18-50 years
• Sex, Male and Female
• Informed Consent, The capability and willingness to give written informed consent to understand the exclusion criteria and to accept the randomized group assignment is required.
• PAR-Q+, The capability and willingness to complete the physical activity readiness questionnaire, with no medical clearance needed.
• Physical Activity: Participants must refrain from regular HIIT and structured breathing practices for a month before participation
• Depression, Anxiety, and Stress Scale, Subcategory Scoring (DASS-21) Participants must score in the mild or greater category for Depression, Anxiety, and Stress. Depression ≥ 10, Anxiety ≥ 8, and Stress ≥ 14.

Exclusion Criteria:

• Age: Individuals under 18 years and over 50 years
• Significant Respiratory Conditions, including but not limited to asthma, chronic obstructive pulmonary disease,
• Significant Cardiovascular Disease: Participants with a known cardiovascular condition such as a previous myocardial infarction, congestive heart failure, stroke, or transient ischemic attack, cardiomyopathy, serious arrhythmia, peripheral vascular disease, untreated atherosclerosis, or hypertension.
• Significant Musculoskeletal Disease, including but not limited to osteopenia, osteoporosis, rheumatoid arthritis, and sarcopenia
• Cognitive and Mental Health, including but not limited to untreated anxiety, untreated depression, post-traumatic stress disorder, and thoughts of suicide.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Health Services Research
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
No Intervention: CON, control; The control group will undergo the same testing procedures outlined above, but they will be asked to maintain their current level of activity and normal daily habits for the duration of the intervention.
Active Comparator: HIG, High intensity group; During the exercise session, HR will be measured via a Polar heart rate strap (Kempele, Finland). The training sessions will consist of six 90-second high-intensity cycling (HIC) sprints performed on a cycle ergometer (Monark Ergomedic 895E, Monark, Varberg, Sweden) at 80-90% VO2max, followed by 180 seconds of low-intensity cycling (LIC) at 50-60% of VO2max. During both HIC and LIC intensities, participants will be asked rate of perceived exertion, which will be compared to the participants' heart rate throughout the exercise sessions. This supervised training protocol will require 9 minutes of HIC at 80-90% VO2max and 18 minutes of LIC at 50-60% VO2max. The cycling session will begin with a brief warm-up and end with a cool-down down totaling the entire supervised exercise session for 30 minutes. Training sessions will be performed 48 hours after the previous training session for a total of 3 times per week on Mondays, Wednesdays, and Fridays.	Behavioral: Hight intensity interval training; Participants assigned to this intervention group will complete 6 supervised HIIT sessions over 2 weeks.; Other Names: HIIT; SIT; Sprint interval training
Active Comparator: CHG, Cyclic hyperventilation group; The breathing intervention will consist of a daily breathing practice lasting 5 to 10 minutes for 5 days per week. Breathing practice is a form of cyclic hyperventilation consisting of 30 breath (inhalation and exhalation) repetitions followed by an exhaled breath retention for 15 seconds during the first week and up to 30 seconds during the second week. A total of 3 rounds will be performed by the participants. Participants will be instructed to perform breathing repetitions in a controlled and consistent manner while either seated or lying down. Participants will receive guided instruction via a video on breath cycle queues, informing the participants when to inhale, exhale, and when to retain their breath while being supervised by the research team via Zoom call. Upon completion of the breath training sessions, the research team will conduct a guided cooldown and check in with all participants to ensure they are feeling no adverse side effects from the breathing exercise.	Behavioral: Cyclic Hyperventilation with Retention; Participants of this intervention group will complete a total of 10 remote guided breathing sessions on Zoom over 2 weeks.; Other Names: CHR

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Heart Rate Variability (HRV)	Heart rate variability (HRV) will be analyzed to determine the fluctuation in the time intervals between adjacent heartbeats to assess autonomic regulation of the heart. Resting HR and HRV were measured using the electrocardiograph (GE CASE Exercise Testing System Version 6.0, Chicago, Illinois). To measure HRV, the standard deviation of normal to normal beats (SDNN) was used with the 10-second electrocardiogram reading. Based on the outcome of this assessment, participants with SDNN values below 50 ms will be classified as unhealthy, 50-100 ms will be classified as having compromised health, and above 100 ms will be classified as healthy.	Visit 2 (week 1) and Vist 4 (week 4)
Serum Analysis: C-reactive protein (CRP)	Approximately 60 mL of whole blood will be collected into serum separator tubes (Beckton Dickinson, East Rutherford, NJ, USA), allowed to clot for 30-min at room temperature, and then centrifuged at 2000 RPM for 15 min. The serum will be pipetted into 1.5 mL microcentrifuge tubes (Eppendorf AG, Hamburg, Germany) and immediately stored in a -80 °C freezer. Serum concentrations of CRP will be determined with a commercially available enzyme-linked immunosorbent assay (ALPCO Diagnostics, Salem, NH, USA). Microplates will be read with an ELx800 BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at the recommended wavelength of 450 nanometers.	Visit 1(Week 1) and Visit 3 (Week 4): 15 minutes for blood draw. The assay itself takes 3 hours.
Serum Analysis: Brain Derived Neurotrophic Factor (BDNF)	Approximately 40 mL of whole blood will be collected into serum separator tubes (Beckton Dickinson, East Rutherford, NJ, USA), allowed to clot for 30 minutes at room temperature, and then centrifuged at 2000 RPM for 15 min. The serum will be pipetted into 1.5 mL microcentrifuge tubes (Eppendorf AG, Hamburg, Germany) and immediately stored in a -80 °C freezer. Serum concentrations of BDNF (Sigma-Aldrich, St. Louis, MO, USA) will be determined with commercially available enzyme-linked immunosorbent assays. Microplates will be read with an ELx800 BioTek microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) at the recommended wavelength of 450 nanometers.	Visit 1A (Week 1) and Visit 2A (Week 4): 15 minutes for blood draw. The assay itself takes 3 hours.
Natural Killer cell quantity	15mL of whole blood will be obtained using sodium heparin tubes and a peripheral venous puncture. Blood samples will be mixed with Dulbecco's phosphate buffer solution (DPBS) at a 1:1 ratio and layered over 20 mL of Ficoll gradient matrix. Blood samples will be centrifugation at 111 x g for 40 minutes, acceleration 7, brake 0 (Eppendorf 5810R centrifuge, Hamburg, Germany). Samples will be washed 3 times, then resuspended at 1.0 * 10^6 cells/mL and incubated for 4 hours at 37 degrees Celsius, with 5% CO2. Next, samples will be pelleted, resuspended, and antibody-receiving samples will each receive 500uL DPBS and 1uL of Fragment crystabillize blocker (FcB)/1.0*10^6 cells and incubate on ice for 20 minutes. Samples will be pelleted, resuspended in 500uL DPBS and receive 500uL of DPBS and 2uL of CD56 (NCAM) monoclonal antibodies, incubate on ice for 20 minutes, pelleted, and resuspended in 1mL of DPBS, then analyzed using an Attune NXT flow cytometer (Thermo Fischer, Waltham, MA).	Visit 1(Week 1) and Visit 3 (Week 4): 15 minutes for blood draw. The assay itself takes ~10-12 hours.
Natural Killer Cell Cytotoxicity	Chronic human myelogenous leukemia cells (K562; ATCC, Manassas, VA) are used to evaluate NKC effector function. K562 cells are stained with Alexa-Fluor 647-conjugated anti-CD71. K562 cells are further stained with Calcein-AM viability dye (Thermo Fisher Scientific, Waltham, MA), then incubated on ice for 20 min. K562 cells are washed and resuspended at 1.0 × 106 cells/mL in complete RPMI 1640 medium. Human PBMCs containing effector NK and NKT cells are co-cultured with K562 cells in 5 mL Eppendorf tubes at a 40:1 PBMC: K562 effector: target (E: T) ratio and incubated for 4 h at 37 °C in a cell incubator. Following co-incubation, samples are centrifuged at 400 x g for 5 min and resuspended in ice-cold DPBS for analysis. After the 4-hour co-culture, these samples are analyzed via flow cytometry to determine the total proportion of alive (Calcein-AM+/CD71+) versus estimated K562 cells. Changes in proportion and MFI of these populations were further analyzed using Floreada.io.	Visit 1(Week 1) and Visit 3 (Week 4): 15 minutes for blood draw. The assay itself takes ~10-12 hours.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Physical Activity Readiness Questionnaire for Everyone (PAR-Q+)	PAR-Q+ utilizes yes or no generalized health questions to screen participants interested in engaging in an exercise program for the need of medical clearance and physician release. The PAR-Q+ has 7 general health questions ranging from Does the participant feel chest pain at rest or during activities of daily living? Individuals who are eligible for this investigation require no further medical clearance before participating in an exercise program and should truthfully answer NO to every question. Participants who answer "Yes" to any questions will be required to answer follow-up questions to determine the need for medical clearance. If medical clearance is suggested, the individual will not be eligible to participate.	Visit 1 (week 1) 10 minutes
Depression Anxiety and Stress Scale (DASS-21)	The DASS-21 contains 3 subscales assessing Depression, Anxiety, and Stress consisting of 7 questions for each subscale, where a high score of 28+ indicates "Extremely Severe" levels of Depression, a high score of 20+ indicates "Extremely Severe" levels of Anxiety, and a high score of 34+ indicates "Extremely Severe" levels of Stress, while low scores of 0-9 indicates "Normal" levels of Depression, scores of 0-7 indicates "Normal" levels of Anxiety, and scores of 0-14 indicates "Normal" levels of Stress. Raw scores for each subcategory will be recorded and multiplied by 2 to calculate the subcategory score.	Visit 1 (week 1) and Visit 3 (week 4) 10 minutes each
Pittsburg Sleep Quality Index (PSQI)	Participants will be asked to complete a paper version of the Pittsburgh Sleep Quality Index (PSQI). The PSQI is a 19-question self-reported sleep quality tool divided into 7 subcategories for scoring: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. Five additional questions directed to the respondent's roommate or bed partner are not used in the scoring but are utilized for clinical purposes.	Visit 1 (week 1) and Visit 3 (week 4) 10 minutes each
Wisconsin Upper Respiratory Symptom Survey (WURSS-21)	The WURSS is an illness-specific quality of life measurement designed to assess the impact of the common cold (acute upper respiratory infection). It consists of 21 questions that investigate the perception of sickness, the severity of sickness-related symptoms over the last 24 hours, the extent to which sickness has hindered the ability to think clearly and accomplish daily tasks over the past 24 hours, and the comparison of sickness to the day before.	Visit 1 (week 1) and Visit 3 (week 4) 10 minutes each
Physical Activity and Sleep Tracking	Heart rate, sleep duration, and steps will be measured and obtained via the WHOOP Strap 4. Measures obtained from the WHOOP strap include continuous heart rate monitoring, sleep cycle, sleep stage, and total step data. The HRV measurement from the WHOOP strap will then be compared to the HRV analysis performed using the standard deviation of the normal-to-normal beat method.	4 weeks
VO2Max	Maximal oxygen consumption will be assessed on a cycle ergometer using a TrueOne 2400 Metabolic Measurement System. A Polar Heart Rate Sensor H1 will be outfitted for each participant before starting the assessment. Participants will rest for 5 minutes in a seated position when heart rate, blood pressure, and blood lactate are measured. Next, the participant will be fitted to the cycle ergometer and perform a brief 5-minute warm-up at 0.5 kg and 60 rpm. Next, the pedal cadence increases to 75-85 rpm, and the participant starts stage 1. Every stage lasts 3 minutes, at the end of every stage, heart rate, blood lactate, blood pressure, and RPE are recorded, and 0.5 kg of resistance is added to the weight basket. Once blood lactate concentration is above 4.0 mmol/L, an incremental increase of 0.2 kg occurs. Assessment is terminated after volitional fatigue, failure to maintain pedal cadence, failure to increase heart rate or VO2 in subsequent stages, and the participant's request.	Visit 2 (week 1) 30-45 minutes

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Anthropometric Measures: Height	Height will be obtained in centimeters (cm)( using a stadiometer SECA 220 (Chino, California, USA).	Visit 1 (week 1)
Anthropometric Measures: Weight	Weight will be obtained in kilograms (kg) using a Detecto standing digital scale (Webb City, Missouri, USA).	Visit 1 (week 1)
Anthropometric Measures: Lean Body Mass	Lean body mass (LBM) will be measured in kilograms and evaluated using air displacement plethysmography using a calibrated BOD POD (COSMED USA Inc., Concord, CA).	Visit 1 (week 1)
Anthropometric Measures: Body Fat Percentage	Body fat percentage (BF%) will be measured as a percentage of estimated body fat relative to total body mass and evaluated using air displacement plethysmography using a calibrated BOD POD (COSMED USA Inc., Concord, CA).	Visit 1 (week 1)
Hydration	A refractometer (Atago, Tokyo, JPN) will be used to evaluate urine-specific gravity with a dehydration cutoff point set to ≥1.020 mg/dL. Dehydrated participants will be given time to hydrate. If dehydration persists, participants will be asked to reschedule the session. After verbal compliance and hydration status are confirmed, participants will proceed with the study's procedures.	Visit 1, Visit 2 (week 1) and Visit 3 (Week 4)

Collaborators and Investigators

• Sponsor: University of Northern Colorado

Study record dates

Study Major Dates

• Study Start (Actual): January 27, 2025
• Primary Completion (Estimated): May 1, 2025
• Study Completion (Estimated): June 1, 2025

Study Registration Dates

• First Submitted: February 11, 2025
• First Submitted That Met QC Criteria: February 11, 2025
• First Posted (Actual): February 17, 2025

Study Record Updates

• Last Update Posted (Actual): April 25, 2025
• Last Update Submitted That Met QC Criteria: April 22, 2025
• Last Verified: January 1, 2025

More Information

Terms related to this study

• Keywords: stress; autonomic nervous system; intervention; training; Natural killer cells; heart rate variability (HRV); High intensity interval training (HIIT); innate immune system; Cyclic hyperventilation with retention (CHR); natural killer cell cytotoxicity
• Additional Relevant MeSH Terms: Nervous System Diseases; Autonomic Nervous System Diseases; Primary Dysautonomias
• Other Study ID Numbers: 2404058931

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: We will make all study documents available by reasonable request (Laura.stewart@unco.edu) up to 3 years after data collection is complete.
• IPD Sharing Time Frame: Start: Jan 27, 2025 - Jan 27, 2028
• IPD Sharing Access Criteria: Anyone who contacts Laura.stewart@unco.edu with a reasonable request.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No