This study was funded by the Mayo Clinic Department of Laboratory Medicine and Pathology and other various internal sources.

The protocol was approved by the Mayo Institutional Review Board and conformed to the Declaration of Helsinki. All participants provided written informed consent before participating in the testing described.
1. Calibration and Setup of Metabolic Cart
<ol>
	<li>Flow and volume (pneumotach) calibration 
	NOTE: Specific materials and equipment are listed in the Materials Table.
	<ol>
		<li>Open the pulmonary function and gas exchange software to calibrate for the test.</li>
		<li>Click the &#8220;calibrate&#8221; button to open the calibration window. At the bottom of the calibration window, note the room temperature, barometric pressure, and the humidity &#8212; ensure that these values are accurate using a barometer.</li>
		<li>Insert the umbilical and sample line into the pneumotach and then insert the pneumotach into the 3 L syringe.</li>
		<li>Before starting calibration, click the &#8220;zero flow&#8221; button to ensure that there is no flow going through the syringe.</li>
		<li>Click &#8220;start&#8221; to begin the calibration. Withdraw first, then inject. Repeat 4 more times (5 total), except at different flow rates each time. Maintain constant flow rate for each respective withdrawal/injection, then vary flow rate for the next iteration.</li>
	</ol>
	</li>
	<li>Gas calibration
	<ol>
		<li>Attach sample line from respiratory mass spectrometer/gas analyzer/metabolic cart to the calibration/home port on the system.</li>
		<li>In the pulmonary function software, select the O2/CO2 analyzers tab in the calibrate window. Open the reference (room air 21% O2, 0.04% CO2) and calibration (12% O2 and 5% CO2) gas tanks.</li>
		<li>Select the calibrate button. 
		NOTE: The software will operate the solenoid valves in the metabolic cart system to switch between the reference and calibration gases being sampled at the calibration port. From this, the software will evaluate for any offset, sampling the delay due to sample line length and the 2&#8211;90% response time. A green &#8220;Calibration Successful&#8221; message will appear when calibration is complete.</li>
	</ol>
	</li>
	<li>Final Setup
	<ol>
		<li>Remove the sample line from the gas calibration port and insert it back into the pneumotach. Then attach a mouthpiece onto the pneumotach.</li>
		<li>Wait for the subject to arrive for exercise test.</li>
		<li>Upon arrival of the subject, inform them about the study and what their involvement will be, review the consent form with them and have him/her sign it, and as the visit proceeds continually discuss with them what (s)he will be doing next and provide explanation of the procedures before doing them. 
		NOTE: Inclusion and exclusion criteria will vary based on the purpose of the exercise testing, for this study those recruited were healthy, non-smokers, with no known cardiopulmonary or immune disease and not taking any steroids or immune modulating drugs.</li>
	</ol>
	</li>
</ol>
2. Pulmonary Function Test (PFT)
NOTE: The pulmonary function test methods described are a brief summary of those published by the American Thoracic Society and European Respiratory Society, for additional details please refer to their publications16,17.
<ol>
	<li>Slow vital capacity (SVC) maneuver
	<ol>
		<li>Instruct the subject to be seated with straight back and feet flat on the floor with legs uncrossed.</li>
		<li>Instruct the subject to put his/her mouth around the mouthpiece, and bite down; also fit the subject with a nose clip to seal off the nasal cavity.</li>
		<li>Start the maneuver on the software. When beginning the maneuver, instruct the subject to continue to breathe normally.</li>
		<li>Observe the subject&#8217;s tidal breathing and have him/her continue to breathe while waiting for him/her to reach a stable breathing pattern.</li>
		<li>Instruct the subject to inhale maximally and then exhale slowly. The subject will keep emptying his/her lungs until they can no longer exhale. This point will be apparent by a plateau in the flow tracing. At this point, instruct the subject to take a maximal breath in.</li>
		<li>Stop the measurement and instruct the subject to release the mouthpiece and remove the nose clip to take a break if needed. 
		NOTE: A minimum of three SVCs are performed. To meet American Thoracic Society standards, they must agree within 5% or 150 mL of each other for both the largest VC and inspiratory capacity (IC) values. Up to 4 maneuvers can be performed in order to obtain three that agree16,17.</li>
	</ol>
	</li>
	<li>Forced vital capacity (FVC) maneuver
	<ol>
		<li>Instruct the subject to grasp the mouthpiece and attach the nose clip while remaining in the same seated position.</li>
		<li>Start the maneuver on the software and instruct the subject to continue to breathe normally. Ensure the subject has established a stable breathing pattern with a minimum of four tidal breaths.</li>
		<li>Instruct the subject to inhale fully and rapidly, and then to immediately exhale (pause of &#60; 1 s) as quickly and forcefully as possible.</li>
		<li>Tell the subject/patient to continue to try and blast/push all the air out of his/her lungs reaching a full exhalation while remaining in an upright posture. This is seen as a plateau in the volume-time curve. Ask them to continue trying to exhale for as long as they can; ideally for 6 s.</li>
		<li>Once this is achieved, instruct the subject to take a maximal breath in and stop the maneuver on the software. 
		NOTE: The test should be repeated at least two more times &#8212; ensuring that they agree within 5% or 150 mL of each other for both the two largest FVC and FEV1 values. Up to 8 maneuvers can be performed in order to get two that agree.</li>
	</ol>
	</li>
	<li>Maximum voluntary ventilation (MVV) maneuver 
	NOTE: The goal of this maneuver is to have the subject move as much air as rapidly as they can. They will be coached to try to take as large breaths as possible while still breathing rapidly.
	<ol>
		<li>Instruct the subject to grasp the mouthpiece and attach the nose clip while maintaining the same seated position.</li>
		<li>Start the maneuver on the software. 
		NOTE: A countdown bar at the top will indicate the number of breaths required (usually three) before data collection/measurements begin.</li>
		<li>With one breath to go in the countdown, direct the patient to start breathing deeply and rapidly through the mouthpiece. They will continue this for 12 s.</li>
		<li>Encourage the patient throughout the procedure to breathe deeply and fast. If the subject is unable to continue, stop the test.</li>
		<li>At the end of 12 s, instruct the patient to resume normal breathing. They may feel light headed, so encourage them to sit back and take slow deep breaths. 
		NOTE: For repeatability the test should be performed a minimum of two times and the suggested variability should be less than 20%.</li>
	</ol>
	</li>
</ol>
3. Exercise Tests
<ol>
	<li>Electrode placement
	<ol>
		<li>Prepare the skin for the electrodes by shaving hair away from electrode placement site if present. Rub the site with an alcohol pad and then with an abrasive pad to remove any dead skin cells. 
		NOTE: Electrode can be placed once this is completed, but be sure that the electrode has adequate gel and is not dry.</li>
		<li>Equip the subject with electrodes for a 12-lead electrocardiogram using the following electrodes placement.
		<ol>
			<li>Position limb lead electrodes as follows: RA: right side subclavicular fossa; LA: left side subclavicular fossa; RL: right back just above posterior superior iliac spine; LL: left back just above posterior superior iliac spine</li>
			<li>Position precordial leads as follows: V1: right of sternum in the 4th intercostal space; V2: left of sternum in the 4th intercostal space (in line with V1); V3: left side directly between V2 and V4; V4: left side in the 5th intercostal space at the midclavicular line (typically under breast/nipple); V5: place horizontally with V4 on anterior axillary line (down from the edge of the armpit (anterior axillary fold of the upper arm); V6: place horizontally with V4 and V5 on the middle auxiliary line.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Incremental maximal cycling test &#8212; Visit 1 
	NOTE: Performing a maximal exercise test&#160;comes with risks. The American College of Sports Medicine outlines how to identify individuals who are at higher risk for an adverse event during the test13. Those who would be considered to have significant risk have: known cardiovascular, pulmonary and/or metabolic disease; major symptoms: chest pain, shortness of breath (SOB) at rest or with mild exertion, dizziness or syncope, orthopnea, ankle edema, palpitations or tachycardia, intermittent claudication, known heart murmur, unusual fatigue or SOB with usual activities; or at least two of the cardiovascular disease risks factors: family history of myocardial infarction or sudden death, age (males &#8805; 45, women &#8805; 55), current smoker, sedentary lifestyle ( &#60; 30 min of moderate intensity physical activity 3 days a week for at least 3 months), obesity (BMI &#8805; 30 kg/m2), hypertension (Systolic blood pressure (SBP) &#8805; 140 mmHg or Diastolic blood pressure (DBP) &#8805; 90 mmHg), dyslipidemia (total cholesterol &#8805; 200 mg/dL; LDL &#8805; 130 mg/dL; HDL &#60; 40 mg/dL, or on lipid lowering medication), prediabetes (fasting blood glucose &#62; 100 mg/dL). All maximal exercise testing should be performed under the supervision of a health care professional trained in clinical exercise testing, with at least two people present, one to monitor the ECG and the other to be taking blood pressures and monitoring the patient. For those who are higher risk, a physician should also be present during the test, whereas in those that are at lower risk the test can be performed without a physician present; it is preferred to have the physician nearby and available immediately if needed. The personnel performing the test should have basic life support with an automated external defibrillator (AED) in the room and at least one or more performing personnel should have advance cardiac life support training. Those performing the test should know the plan for responding to a medical emergency and have the appropriate contact numbers.
	<ol>
		<li>Fit the subject to the bike making sure the seat and handlebars are positioned comfortably. 
		NOTE: The general suggestion for seat height is such that the leg has a slight bend at the bottom of the pedal stroke and the seat should be adjusted horizontally so that when the crank arm is parallel to the ground the knee should fall over the metatarsals which should be over the pedal spindle. The handlebars should be at the same height as the seat or slightly higher and close enough so that the subject has a slight bend in their elbows. The handlebar position will depend on the experience of the rider, more experienced cyclists will want to be more bent over, where are those who do not bike often will prefer a more upright position.</li>
		<li>Place a pulse oximeter on the subject&#8217;s forehead. Wipe the placement location with an alcohol wipe to remove any makeup or dirt, etc. and secure it to the forehead with a headband.</li>
		<li>Review the exercise test procedure with the subject. Inform the subject that they will need to stay breathing through the mouthpiece for the entire duration of the test, and breathe only through his/her mouth as the nose will be plugged with the nose clip. 
		NOTE: Using a mouthpiece and nose clip is not the only option; masks are available which cover the nose and mouth allowing the participant to breathe through either their mouth or nose18. Gas exchange metrics are continuously measured and recorded through the pulmonary function software. Heart rate (HR) and rhythm will be continuously monitored by the 12-lead electrocardiogram (ECG). Peripheral oxygen saturation (SpO2) will be continuously monitored with the pulse oximeter. These external signals (HR, SpO2) can be linked to the metabolic cart so that all measurements are documented together. If this isn&#8217;t possible, HR and SpO2 should be recorded on a worksheet every minute.</li>
		<li>Draw a 5 mL blood sample from the antecubital vein (baseline exercise blood draw).</li>
		<li>After 2 min of rest, begin data collection. Then, start the exercise protocol and instruct the participant to start&#160;pedaling. Ask them to reach a pedal rate between 60 and 80 rpms. 
		NOTE: For this study, the maximal exercise test protocol used was an initial workload of 50 W with 30 W increments every 2 min. The protocol used can vary depending upon the population and the goals of the test. For older individuals or patient populations, the first stage can be performed unloaded to allow the individuals to get their legs moving before resistance is added. In younger healthy individuals, this is usually not necessary as 50 W is a low enough workload to warm up. Pedaling at 0 W while maintaining the desired pedal rate is actually more challenging than starting with resistance from the beginning.</li>
		<li>Have an assisting technician measure blood pressure (BP) 1 min into each stage while a second technician assists with the test. Then ask the subject to rate his/her exertion level on the Borg Rating of Perceived Exertion (RPE) scale where 6 indicates that the exertion is perceived as easy (like (s)he is sitting/standing doing nothing) and 20 indicates that the perceived exertion is at the hardest work they can imagine doing18. Take a 12-lead ECG printout within the last 30 seconds of each stage.</li>
		<li>Continue the test until exhaustion of subject which is designated by having at least two of the following occur: when 60-80 rpm on the bike can no longer be maintained, the subject&#8217;s VO2 plateaus and does not increase with an increase in workload, his/her respiratory exchange ratio (RER) is equal to or greater than 1.1&#8211;1.2, and/or the subjects rating of perceived exertion (RPE) &#8805; 18.</li>
		<li>Stop the test if any of the following occur13: onset of angina or chest pain symptoms; drop in SBP of &#8805; 10 mmHg with an increase in work; excessive rise in BP: SBP &#62; 250 mmHg and/or DBP &#62; 115 mmHg; shortness of breath, wheezing, leg cramps, or claudication; signs of poor perfusion: light-headedness, confusion, nausea, cyanosis, cold or clammy skin; failure of HR to increase with increasing exercise intensity; change in heart rhythm with symptoms; subject requests to stop; subject vocalizes or severe fatigue is observed; testing equipment not functioning properly.</li>
		<li>Upon reaching exhaustion, proceed to the recovery phase: drop the resistance to the initial workload and instruct the subject to continue to cycle for another 2 min.</li>
		<li>Draw another 5 mL of blood from the patient (post exercise blood draw) via antecubital venipuncture.</li>
		<li>Direct the subject to return in 3 h, and 24 h following the completion of the test for the additional 5 mL blood draws. Instruct the subject to not partake in further exercise until completion of the 3rd post exercise blood draw at 24 h. 
		NOTE: These were the time points chosen for this study to evaluate the time line of changes in immune cells. The questions and parameters of interest will dictate when sampling should occur.</li>
	</ol>
	</li>
	<li>Endurance steady-state submaximal cycling test &#8212; Visit 2
	<ol>
		<li>Complete steps 1 for calibration, 3.1 for ECG preparation, 3.2.1 for bike fitting and 3.2.2 for forehead pulse oximeter installation.</li>
		<li>Review the exercise test procedure with the subject. Inform the subject that they will be biking for 45 min, but unlike the maximal exercise test, they will only be asked to breathe through the mouthpiece with the nose clip for sections of test and not continuously. 
		NOTE: As with the maximal exercise test, HR and rhythm will be continuously monitored by the 12-lead ECG. SpO2 will be continuously monitored with the pulse oximeter.</li>
		<li>Draw 5 mL of venous blood sample from the antecubital vein (baseline endurance submaximal exercise blood draw) before starting the test.</li>
		<li>Instruct the subject to grasp the mouthpiece by mouth and attach the nose clip.</li>
		<li>Begin data collection, and then start the exercise protocol. Instruct the participant to start pedaling, and ask them to reach a pedal rate between 60 and 80 rpms. 
		NOTE: For this study the endurance steady-state protocol is 45 min with a 3&#8211;5 min warm up at 50 W. Following warmup, the wattage is increased to 60% of the subject&#8217;s maximal workload determined from visit 1. This is different from visit 1 in that the workload is constant and the bout is set for a specific duration, rather than increasing the workload until peak VO2 is reached.</li>
		<li>Measure BP (use the same technician as before) and ask the subject to report exertion level intermittently (every 3&#8211;5 min) throughout the exercise.</li>
		<li>Instruct the subject to release the mouthpiece from the 10th min to 25th min, and re-grasp the mouthpiece from the 25th min to 30th min and during last 5 min of 45 min bout (40th min to 45th min). 
		NOTE: Gas exchange metrics are being intermittently monitored only when the subject is on the mouthpiece; intermittent monitoring is done as the mouthpiece can be dry and uncomfortable for the subject when used for longer periods of time. Since the goal of this type of exercise test is to have the subject reach specified exercise intensity and then hold that as a steady-state, the subject&#8217;s gas exchange does not need to be continuously monitored unless it is a primary outcome measure. In the case of this study it was the stimulus, and not the outcome of interest.</li>
		<li>Ensure that the test remains at a steady-state by monitoring the following metrics:
		<ol>
			<li>Check that the VO2 hasn&#8217;t increased significantly (+/- 5 mL/min/kg) when the subject is back on the mouthpiece.</li>
			<li>Check that the subject&#8217;s HR doesn&#8217;t increase by more than 5 bpm.</li>
			<li>Observe for the subject appearing tired or that his/her RPE rating is rising.</li>
		</ol>
		</li>
		<li>Drop the workload by ~5&#8211;10% to ensure completion of 45 min of cycling, if any of the above occurs.</li>
		<li>Following 45 min, instruct the participant to complete a 2 min recovery period of easy pedaling, and then draw 5 mL of venous blood (post exercise blood draw).</li>
		<li>Give the same post exercise instructions as in visit 1 i.e., do not engage in exercise until after the 24 h time point and return to the lab for blood draws 3 h and 24 h post exercise.</li>
	</ol>
	</li>
</ol>
4. Blood Analysis
<ol>
	<li>Process blood samples for analysis. 
	NOTE: Potential methods may include, but are not limited to, flow cytometry of circulating leukocytes, cytokine analysis of plasma samples, and/or leukocyte gene expression analysis. Additionally, optimal time points may need to be empirically determined.</li>
</ol>

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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=General+considerations+for+lung+function+testing.">General considerations for lung function testing.</a> European Respiratory Journal. 26 (1), 153-161 (2005).</li><li>Borg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratings+of+perceived+exertion+and+heart+rates+during+short-term+cycle+exercise+and+their+use+in+a+new+cycling+strength+test.">Ratings of perceived exertion and heart rates during short-term cycle exercise and their use in a new cycling strength test.</a> International Journal of Sports Medicine. 3 (3), 153-158 (1982).</li><li>Norton, K., Norton, L., Sadgrove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position+statement+on+physical+activity+and+exercise+intensity+terminology.">Position statement on physical activity and exercise intensity terminology.</a> Journal of Science and Medicine in Sport. 13 (5), 496-502 (2010).</li><li>Lansley, K. E., Dimenna, F. J., Bailey, S. J., Jones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+'new'+method+to+normalise+exercise+intensity.">A 'new' method to normalise exercise intensity.</a> International Journal of Sports Medicine. 32 (7), 535-541 (2011).</li><li>Poole, D. C., Burnley, M., Vanhatalo, A., Rossiter, H. B., Jones, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+Power:+An+Important+Fatigue+Threshold+in+Exercise+Physiology.">Critical Power: An Important Fatigue Threshold in Exercise Physiology.</a> Medicine and Science in Sports and Exercise. 48 (11), 2320-2334 (2016).</li><li>Gustafsson, 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=Effects+of+Acute+Exercise+on+Circulating+Soluble+Form+of+the+Urokinase+Receptor+in+Patients+With+Major+Depressive+Disorder.">Effects of Acute Exercise on Circulating Soluble Form of the Urokinase Receptor in Patients With Major Depressive Disorder.</a> Biomarker Insights. 12, 1177271917704193 (2017).</li><li>Hallberg, L., 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=Exercise-induced+release+of+cytokines+in+patients+with+major+depressive+disorder.">Exercise-induced release of cytokines in patients with major depressive disorder.</a> Journal of Affective Disorders. 126 (1-2), 262-267 (2010).</li><li>Bengtsson Lindberg, M., Wilke, L., Vestberg, S., Jacobsson, H., Wis&#233;n, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exercise-induced+Release+of+Cytokines/Myokines+in+a+Single+Exercise+Test+before+and+after+a+Training+Intervention+in+Patients+with+Mild+Cognitive+Impairment.">Exercise-induced Release of Cytokines/Myokines in a Single Exercise Test before and after a Training Intervention in Patients with Mild Cognitive Impairment.</a> International Journal of Physical Therapy &amp; Rehabilitation. 3, (2017).</li></ol>

The authors declare that they have no competing financial interests.

There is great potential for exercise to be incorporated as an adjunct/alternative therapeutic tool. Indeed, an emerging body of evidence strongly suggests that physical activity promotes good health. The use of exercise as a medicine or diagnostic tool would require an understanding of the right amount or &#34;dose&#34; of exercise to achieve the desired effect. The optimal dose of exercise should be estimated, as too much exercise may be detrimental to improving health. As such, an exercise regimen may need to be tailo...

Physical activity is defined as any bodily movement produced by skeletal muscles that require energy expenditure1. Exercise is a physical activity that involves repetitive bodily movement done to improve or maintain one or more components of physical health2. At one time, physical activity was not recommended for those who were seriously ill. For individuals with cancer, heart failure, or even for those who were pregnant, bed rest was preferred over physical activity. Clinical practice has since drastically changed, as the benefits of exercise on overall health are becoming undeniable3. Regular exercise has been shown to help reduce cardiovascular disease risk, all-cause mortality, cancer risk and hypertension, improve blood sugar control, facilitate weight loss or maintenance, and prevent bone and muscle loss4,5,6,7,8.
The extensive benefits of exercise have now led many to utilize exercise as a type of &#34;medicine&#34; and an alternative or adjunct treatment option for a variety of conditions3. Shulman et al. demonstrated that a combination of treadmill and resistance exercise could result in improvements in gait speed, aerobic capacity and muscular strength which could improve motor control and overall quality of life in patients with Parkinson&#39;s disease9. In heart failure patients, exercise intolerance and inadequate pharmaceutical interventions contribute to a poor quality of life10. Initial results from heart failure patients undergoing exercise training in the HF-ACTION trial demonstrated improvement in quality of life and reductions in hospitalizations and mortality11. Additionally, the application of exercise to alter the cardiotoxic effects of anthracycline-containing chemotherapy (e.g., doxorubicin) has demonstrated that regardless of when it is initiated with respect to the patients chemotherapy administration (before, during or after), exercise can provide beneficial effects such as reducing the decline in aerobic capacity, attenuating the left ventricular dysfunction and reducing oxidative damage12.
The benefits of exercise in health and wellness are not just in its application as a medicine/treatment, but also as a diagnostic tool. Exercise testing is, for example, used to diagnose exercise intolerance, ischemia in the heart, or to understand the cause of shortness of breath13. Perhaps more importantly, exercise testing may be utilized to identify subclinical dysfunction. The human body is in most situations &#34;overbuilt,&#34; such that dysfunction or pathophysiology can often remain hidden and unapparent to an individual for months or years. This observation may explain why conditions such as pulmonary arterial hypertension or pancreatic cancer can silently increase in severity such that by the time symptoms are noticed,&#160;these conditions tend to be very advanced and extremely difficult to treat2. In some of these situations, exercise testing can provide a stress stimulus to the body which increases demand above that of daily living and at times can identify dysfunction (cardiac, respiratory, metabolic) that was not seen at rest, helping to diagnose a disease and begin treatment earlier.
In order to fully maximize the therapeutic and diagnostic potential of exercise, standardized methods to quantify the responses to physical activity are needed to accurately assess the contributions of exercise to overall immune health. Variations in workload, inclination, duration, type of exercise, and the timing of sample collection can all influence measurements of physiological responses. Here, we outline methods for maximal and submaximal endurance exercises to gather physiological data while collecting samples for biological responses. This methodology was used to understand how acute exercise affected the distribution and frequency of leukocyte populations in peripheral blood14 by measuring&#160;immune cell populations at various time points before and after exercise by flow cytometry with 10-color flow protocols that permit the quantification of all major leukocyte subsets simultaneously15. The following protocol can be used as a standardized method for two distinct exercise regimens for measuring physiological and biological responses to exercise.

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Metabolic cart/portable system</td>
			<td style="width:121px;">MCG Diagnostics</td>
			<td style="width:181px;">Mobile Ultima CPX System</td>
			<td style="width:323px;">The flow calibration syringe, and calibration gases should come with system. There are numerous possible options/alternatives.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pulmonary function software (Breeze Suite)</td>
			<td>MCG Diagnostics</td>
			<td></td>
			<td style="width:323px;">Software used will depend on the metabolic system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Upright cycle ergometer</td>
			<td>Lode ergoline</td>
			<td>960900</td>
			<td>Numerous possible options/alternatives</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;">12-Lead ECG</td>
			<td>GE Healthcare</td>
			<td style="width:181px;">CASE Exercise Testing System</td>
			<td style="width:323px;">Used for 12 lead ECG capture, control bike. Having a full 12-lead is ideal for maximal exercise test so can monitor for arhythmias, but alternative for just HR would be a wireless chest strap heart rate monitor</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Pulse oximeter</td>
			<td>Masimo</td>
			<td>MAS-9500</td>
			<td style="width:323px;">Usually multiple probe options: finger, forehead, ear lobe.&#160; Usually avoid finger as tight handlebar grip can cause measurement inaccuracies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pneumotach (preVent Flow Sensor)</td>
			<td>MCG Diagnostics</td>
			<td>758100-003</td>
			<td>Alternative systems can use a turbine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nose piece (disposable)</td>
			<td>MCG Diagnostics</td>
			<td>536007-001&#160;</td>
			<td>Numerous possible options/alternatives</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mouthpeice with saliva trap</td>
			<td>MCG Diagnostics</td>
			<td>758301-001</td>
			<td style="width:323px;">Suggest filling the saliva trap with paper towel/gauze and tape cap to limit dripping</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Headband</td>
			<td>Cardinal Health</td>
			<td>292866</td>
			<td style="width:323px;">Used to secure the forehead pulse oximeter and the lines for the cart</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stethescope&#160;</td>
			<td>3M Littman</td>
			<td>3157SM</td>
			<td>Numerous possible options/alternatives</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Blood pressure cuff</td>
			<td>HCS</td>
			<td>HCS9005-7</td>
			<td style="width:323px;">Cuff size will depend on the population planning to test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ECG Electrodes</td>
			<td>Cardinal Health</td>
			<td>M2570</td>
			<td>only needed with lead based ECG/HR monitoring</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">K2EDTA tube 5mL</td>
			<td>Becton Dickinson</td>
			<td>368661</td>
			<td></td>
		</tr>
		<tr height="79">
			<td colspan="4" height="79" style="height:79px;width:841px;">*The table provides a list of the supplies and equipment utilized in this protocol and comments related to the equipment. Brand name/company is provided, but the use of other brands will not affect the results, key is to keep it consistent throughout testing in a particular study.</td>
		</tr>
	</tbody>
</table>

conducting maximal and submaximal endurance exercise testing to measure physiological and biological responses to acute exercise in humans

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic responses to acute exercise are predominantly influenced by the duration and intensity of the exercise regimen. As exercise is increasingly thought of as a therapeutic treatment and/or diagnostic tool, it is important that standardizable methodologies be utilized to understand the variability and to increase the reproducibility of exercise outputs and measurements of responses to such regimens. To that end, we describe two different cycling exercise regimens that yield different physiologic outputs. In a maximal exercise test, exercise intensity is continually increased with a greater workload resulting in an increasing cardiopulmonary and metabolic response (heart rate, stroke volume, ventilation, oxygen consumption and carbon dioxide production). In contrast, during endurance exercise tests, the demand is increased from that at rest, but is raised to a fixed submaximal exercise intensity resulting in a cardiopulmonary and metabolic response that typically plateaus. Along with the protocols, we provide suggestions on measuring physiologic outputs that include, but are not limited to, heart rate, slow and forced vital capacity, gas exchange metrics, and blood pressure to enable the comparison of exercise outputs between studies. Biospecimens can then be sampled to assess cellular, protein, and/or gene expression responses. Overall, this approach can be easily adapted into both short- and long-term effects of two distinct exercise regimens.

The application of maximal or submaximal endurance exercise testing provides a stimulus or stressor in which the body responds to meet the increased physiological demands. Various modes of exercise can be used to compare the physiological and biological responses to a particular exercise by itself or when a drug/intervention is used, or to evaluate the differences in responses between different exercise loads. Maximal and endurance exercise loads differ in the duration (short/long respect...

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Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Regular physical activity has a positive effect on human health, but the mechanisms controlling these effects remain unclear. The physiologic and biologic ...

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20.1K Views.  Mayo Clinic. To assess the influence of exercise intensity on physiologic and biologic responses, two different exercise testing protocols were utilized. Methods outlining exercise testing on a cycle ergometer as an incremental maximal oxygen consumption test and endurance, steady state submaximal endurance test are described.

Video: Conducting Maximal and Submaximal Endurance Exercise Testing to Measure Physiological and Biological Responses to Acute Exercise in Humans

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Using Continuous Data Tracking Technology to Study Exercise Adherence in Pulmonary Rehabilitation

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to exercise training recommendations. The study of exercise adherence is thus a key step towards the optimization of PR programs. To date, mostly indirect measures, such as rates of participation, completion, and attendance, have been used to determine adherence to PR. The purpose of the present protocol is to describe how continuous data tracking technology can be used to measure adherence to a prescribed aerobic training intensity on a second-by-second basis.
In our investigations, adherence has been defined as the percent time spent within a specified target heart rate range. As such, using a combination of hardware and software, heart rate is measured, tracked, and recorded during cycling second-by-second for each participant, for each exercise session. Using statistical software, the data is subsequently extracted and analyzed. The same protocol can be applied to determine adherence to other measures of exercise intensity, such as time spent at a specified wattage, level, or speed on the cycle ergometer. Furthermore, the hardware and software is also available to measure adherence to other modes of training, such as the treadmill, elliptical, stepper, and arm ergometer. The present protocol, therefore, has a vast applicability to directly measure adherence to aerobic exercise.

Pulmonary rehabilitation is widely recognized in the management of respiratory diseases. A key component to successful pulmonary rehabilitation is adherence to the recommended exercise training. The purpose of the present protocol is to describe how continuous data tracking technology can be used to precisely measure adherence to a prescribed aerobic training intensity.

Pulmonary rehabilitation (PR) is an important component in the management of respiratory diseases. The effectiveness of PR is dependent upon adherence to ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

A Methodological Approach to Non-invasive Assessments of Vascular Function and Morphology

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis. Damage to the endothelium may predispose the vessel to atherosclerosis and increase the risk for cardiovascular disease. Assessments of peripheral endothelial function are good indicators of early abnormalities in the vascular wall and correlate well with assessments of coronary endothelial function. The present manuscript details the important methodological steps necessary for the assessment of microvascular endothelial function using laser Doppler imaging with iontophoresis, large vessel endothelial function using flow-mediated dilatation, and carotid atherosclerosis using carotid artery ultrasound. A discussion on the methodological considerations for each of the techniques is also presented, and recommendations are made for future research.

The present article describes the methodological considerations for several non-invasive assessments of vascular function and morphology that are commonly used in medical research to assess different stages of atherosclerosis.

The endothelium is the innermost lining of the vasculature and is involved in the maintenance of vascular homeostasis.  Damage to the endothelium may ...

Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies.
Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

We present a biochemical and behavioral protocol to evaluate the efficacy of mitochondria-targeted water-soluble compounds for the treatment of Spinocerebellar ataxia type 1 (SCA1) and other cerebellar neurodegenerative diseases.

Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ...

Surgical Placement of Catheters for Long-term Cardiovascular Exercise Testing in Swine

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early cardiopulmonary dysfunction is difficult to diagnose, particularly in animal models, as cardiopulmonary function is often measured invasively, requiring anesthesia. As many anesthetic agents are cardiodepressive, subtle changes in cardiovascular function may be masked. In contrast, chronic instrumentation allows for measurement of cardiopulmonary function in the awake state, so that measurements can be obtained under quiet resting conditions, without the effects of anesthesia and acute surgical trauma. Furthermore, when animals are properly trained, measurements can also be obtained during graded treadmill exercise.
Flow probes are placed around the aorta or pulmonary artery for measurement of cardiac output and around the left anterior descending coronary artery for measurement of coronary blood flow. Fluid-filled catheters are implanted in the aorta, pulmonary artery, left atrium, left ventricle and right ventricle for pressure measurement and blood sampling. In addition, a 20 G&#160;catheter is positioned in the anterior interventricular vein to allow coronary venous blood sampling.
After a week of recovery, swine are placed on a motor-driven treadmill, the catheters are connected to pressure and flow meters, and swine are subjected to a five-stage progressive exercise protocol, with each stage lasting 3 min. Hemodynamic signals are continuously recorded and blood samples are taken during the last 30 sec of each exercise stage.
The major advantage of studying chronically instrumented animals is that it allows serial assessment of cardiopulmonary function, not only at rest but also during physical stress such as exercise. Moreover, cardiopulmonary function can be assessed repeatedly during disease development and during chronic treatment, thereby increasing statistical power and hence limiting the number of animals required for a study.

Here we present a protocol to assess cardiopulmonary function in awake swine, at rest and during graded treadmill exercise. Chronic instrumentation allows for repeated hemodynamic measurements uninfluenced by cardiodepressive anesthetic agents.

This protocol describes the surgical procedure to chronically instrument swine and the procedure to exercise swine on a motor-driven treadmill. Early ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacy&#160;of a potential therapeutic treatment.

Measurement of rodent skeletal muscle contractile function is a useful tool that can be used to track disease progression as well as efficacy of therapeutic intervention. We describe here the non-invasive, in vivo assessment of the dorsiflexor muscles that can be repeated over time in the same mouse.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively ...

Integration of Brain Tissue Saturation Monitoring in Cardiopulmonary Exercise Testing in Patients with Heart Failure

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction (HF). However, in clinical cardiopulmonary exercise testing (CPET), cerebral hemodynamics is not assessed. NIRS is used to measure cerebral tissue oxygen saturation (SctO2) in the frontal lobe. This method is reliable and valid and has been utilized in several studies. SctO2 is lower during both rest and peak exercise in patients with HF than in healthy controls (66.3 &#177; 13.3% and 63.4 &#177; 13.8% vs. 73.1 &#177; 2.8% and 72 &#177; 3.2%). SctO2 at rest is significantly linearly correlated with peak VO2 (r = 0.602), oxygen uptake efficiency slope (r = 0.501), and brain natriuretic peptide (r = -0.492), all of which are recognized prognostic and disease severity markers, indicating its potential prognostic value. SctO2 is determined mainly by end-tidal CO2 pressure, mean arterial pressure, and hemoglobin in the HF population. This article demonstrates a protocol that integrates SctO2 using NIRS into incremental CPET on a calibrated bicycle ergometer.

This protocol integrated near-infrared spectroscopy into conventional cardiopulmonary exercise testing to identify the involvement of the cerebral hemodynamic response in exercise intolerance in patients with heart failure.

Cerebral hypo-oxygenation during rest or exercise negatively impacts the exercise capacity of patients with heart failure with reduced ejection fraction ...

Isolation of Intrapulmonary Artery and Smooth Muscle Cells to Investigate Vascular Responses

The intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs) isolated from rat lungs can be used to study the underlying mechanisms of vasoconstriction and vasorelaxation. After isolating the IPA and VSMCs, the characteristics of vascular responses in physiological and pathological conditions can be assessed in the absence of extrinsic factors such as nerve signals, hormones, cytokines, etc. Thus, the IPA and VSMCs serve as excellent models for studying vascular physiology/pathophysiology, along with various experimental investigations, such as modulation by pharmacological agents, patch-clamp electrophysiological analysis, calcium imaging, etc. Here, we have used a technique for isolating the IPA to investigate vascular responses in an organ bath setup. IPA segments were mounted on the organ bath chamber via intraluminal wires and stimulated by various pharmacological agents. The changes in IPA vascular tone (i.e., vasoconstriction and vasorelaxation), were recorded using an isometric force transducer and physiological data analysis software program. We implemented several experimental protocols, which can be adapted to investigate the mechanisms of vasorelaxation/vasoconstriction for studying the pharmacological activities of phytochemical or synthetic drugs. The protocols can also be used to evaluate drugs&#39; roles in modulating various diseases, including pulmonary arterial hypertension. The IPA model allows us to investigate the concentration-response curve, which is crucial in assessing drugs&#39; pharmacodynamic parameters.

Vascular responses of arterial pulmonary circulation can be explored using intrapulmonary artery (IPA) and vascular smooth muscle cells (VSMCs). The present study describes the isolation of IPA in detail and the protocols used for investigating vasorelaxation in response to physiological stimuli.