Research primary objective and design
The primary objective of the CAARE study is to determine the differences in the immune response to an acute bout of exercise when considering age and physical activity status of the individual and mode of exercise. CAARE is a randomized, complete cross-over 2 x 2 x 2 (age x physical activity status x mode) factorial design with three timepoints (pre-exercise, post-exercise, and recovery). Apparently healthy young (18-30 years) and older (55-75 years) adults who are currently meeting ACSM exercise recommendations(8) (physically active) or are not exercising (physically inactive) will participate in two moderate to vigorous exercise bouts of different modes: a stacked weight machine resistance exercise bout (RE) and a motor-driven treadmill cardiorespiratory exercise (CRE) bout. Participants are categorized as young/old and active/inactive, then stratified by sex and randomized to which exercise bout they will complete first. We hypothesize that physical activity status will have a bigger influence than age on our variables.
Participant eligibility and recruitment
We are recruiting healthy adults for this study. Participants are recruited from flyers, attendance at health or community fairs, classrooms (on campus and at community centers), and word-of-mouth. To be eligible for participation, potential participants must be classified as “young” and be 18-30 years of age, or “older” and 55-75 years. Females in the “older” age classification must be post-menopausal. Within each age classification, we are recruiting participants who are physically active or inactive. Physically active is defined by the American College of Sports Medicine (ACSM) and American Heart Association primary physical activity recommendations(8) of at least 150 minutes per week of moderate intensity cardiorespiratory exercise training, 60 minutes per week of vigorous intensity cardiorespiratory exercise training, or some combination of moderate and vigorous cardiorespiratory exercise training. In addition to the cardiorespiratory exercise training, the physically active exercise participants must participate in at least two days per week of skeletal muscle strength and endurance training exercises. Physically inactive participants complete less than two days per week of moderate intensity cardiorespiratory exercise training of less than 60 minutes weekly total, no vigorous intensity cardiorespiratory exercise training, and no skeletal muscle strength and endurance training exercises.
Participant exclusion criteria includes: contraindications to moderate or vigorous exercise; recent illness; instructions from a healthcare provider to not exercise; range of motion limitations that would prevent proper exercise form; use of prescription or over the counter medications known to influence immune function (i.e. daily NSAID, statins, bisphosphonates, steroids, etc.); report cardiovascular, respiratory, metabolic, or renal disease, with the exception of controlled hypertension (as defined by resting BP below 140/90); self-reported uncontrolled asthma; gravid; excessive travel that would impact study visit schedule; >30 BMI <18.5 kg·m-2; or unable to refrain from alcohol or recreational drugs for 24h prior to study visits. This study was approved by the institutional review board at the University of Houston (STUDY00001191) and the study setting is the Laboratory of Integrative Physiology In the Department of Health and Human Performance at the University of Houston (academic research laboratory).
Participant enrollment and data collection
After a potential participant expresses interest in the study, they are provided with information to help them determine if they meet self-reported requirements for participation (i.e. a list of exclusionary medications, been instructed by a health care member to not exercise, known cardiometabolic diseases, etc.). If the participant felt they met the basic inclusion/exclusion criteria, they were scheduled for study visit one at the Laboratory of Integrative Physiology (Figure 1). After written informed consent was obtained by a co-investigator, the following measures were obtained: height, weight, blood pressure, body composition, medical history, physical activity frequency, Positive and Negative Affect Schedule (PANAS) and Profile of Mood States Questionnaire (PMSQ), and heart rate variability (HRV). If the participant met all the study qualifications, they were scheduled for visit two. Visit two occurred immediately after visit one or up to fourteen days later and consists of an estimated treadmill 02peak test, strength tests, body composition estimation, and a resistance exercise familiarization session.
Visits three and four are the primary data collection visits. In a random counterbalanced order, participants complete a CRE bout at one visit and a RE bout at the other visit. Participants come to the laboratory after an overnight fast (water ad libitum was allowed and encouraged). After resting in a seated position for 15 minutes, a pre-exercise (PRE) blood sample was collected. The participant completes a five-minute warm-up on a motor-driven treadmill, then performs their assigned exercise bout for the visit. The cardiorespiratory bout consists of 30 minutes of walking or running at 70% of heart rate reserve (HRR). The resistance training bout consists of three sets of eight exercises at 70% 1RM, and takes approximately 30 minutes to complete.
Measurements and assessments
Physical activity assessment and monitoring
After consent, participants are asked to complete an exercise frequency questionnaire. This questionnaire lists commonly performed exercise and physical activities. The participant is asked to estimate pace (if applicable), days per week spent doing the activity, and time per day spent doing the activity. Accelerometers (ActiGraph WGT3X-BT, ActiGraph LLC, Pensacola, FL) are used to objectively measure physical activity. After enrollment, participants are asked to wear the accelerometer on their ankle for one week. The accelerometry data is used to confirm the self-reported physical activity level in the exercise frequency questionnaire.
Body composition estimations are collected with a whole-body dual energy x-ray absorptiometry (Horizon A, Hologic, Marlborough, MA USA). Participants are scanned in the morning after an overnight fast and laying on the table for five minutes.
A two-stage motorized sub-maximal treadmill test is used to estimate 02peak. Each stage is three minutes long, and heart rate is noted in the last 15 seconds of each stage. The participants are asked to walk or run both stages (their preference). The incline is increased for the second stage, and if needed to increase the heart rate >20 BPM the speed is also increased. The 02 is calculated for each stage, and the slope of the line is calculated and used to estimate 02peak.
Skeletal muscle strength
Participants perform an eight-repetition maximum test on each of the eight pieces of equipment used for the resistance exercise study visit. The one repetition maximum (1RM) is estimated from the eight repetition weight(9) , and used to calculate the weight loads on the resistance exercise study visit.
Cardiorespiratory endurance exercise bout
The heart rate reserve (HRR) method is used to calculate CRE exercise study visit intensity. Specifically, the CRE bout is 30 minutes of walking at 60% - 70% HRR on a motor-driven treadmill. The 30-minute bout is preceded by five minutes and followed by two minutes of treadmill walking at a pace self-selected by the study participant. This two-minute cool-down occurs after the POST blood sample is collected from the participant. During the walking bout, HR is monitored to ensure the bout is within the target HR zone (60%-70% HRR). RPE is collected using the 6-20 Borg Scale.
Resistance exercise bout
The RE bout is approximately 30 minutes at 70% of estimated 1RM on eight stacked-weight machines (leg press, leg extension, leg curl, weighted calf raise, chest press, lateral pull down, seated row, and triceps extension). Participants perform three sets of 8- 12 repetitions, with an approximate 30 seconds of rest between sets. All sets at one station are completed before the participant moves to the next station. As with the CRE bout, the bout is preceded by a five minute warm-up walk on the treadmill, and followed by a two minute cool-down walk. Both paces are self-selected by the participant, and the POST blood sample is collected prior to the cool-down.
Blood samples are collected pre-exercise (PRE), post-exercise (POST), and 1hr recovery (RECOV). Sodium heparinized whole blood is used for cell counts, flow cytometry, and LPS stimulation. Serum is separated and stored at -80°C for batch analyses of inflammatory cytokines and acute phase proteins, and biomarkers of metabolism. The main planned variables to be studied in this project are circulating classical (CD14+CD16-) and pro-inflammatory (CD14+CD16+) monocyte percentages, monocyte function, and T-cell subsets.
Heart rate variability
Participants are fitted with a Polar heart rate monitor (Polar V800) and then lay down for five minutes. After this five-minute rest, data is collected for ten minutes while the participant is laying supine. HRV is collected at visit one and after each exercise bout. The post-exercise bout HRV measurement is collected approximately 30 minutes after the end of exercise. R-R intervals will be analyzed with kubios HRV Analysis Software v.1.1 (Biomedical Signal Analysis Group, University of Kuopio, Finland).
Recruitment will be targeted based on the two age groups and physical activity status. Flyers and recruitment tables will be set up at locations to target physically active participants (fitness facilities, fitness clubs, sports clubs, etc.) and physically inactive participants (general health fairs). The older adult group will also be recruited from local senior centers, senior health fairs, and adult education classes. The young adult group will also be recruited from the student body at the University of Houston. Enrollment is anticipated to take two years.
Discontinuing or modifying the protocol
Participation is voluntary, and at any time the participant may request to not continue the study at no consequence to them. The participant may be removed from the study or data analyses upon reporting of new information that changes their responses to the inclusion/exclusion criteria (i.e. reporting of new medication or change in health status, etc.). If the participant is unable to complete the exercise bout at the prescribed intensity, the intensity may be reduced 2-5% so that the participant can complete the bout. Modifications outside this plan will be reported to the institutional review board.
Data analyses plan
For each outcome, we will fit a mixed effects model on the three factors and their interactions. Baseline values will act as a covariate, and the intercept in the change-from-baseline model will be tested to assess post-exercise effects within each physical activity status group and mode of exercise. Prior to model fitting, data will be transformed to improve model fit (i.e., normality, constant within-group variance). As only two time points will be included in the model, no adjustment for autocovariance will be possible. Contrasts will be used to compare individual timepoints across the three factors. Since testing will be done acutely, we assume that data missingness will be rare, largely due to problems with measurement or sample collection, and thus ignorable. Statistical significance will be declared for p-values less than 0.05. Tukey Honest Significant Difference testing will be used to adjust alpha levels to control the Type I error rate. Covariates such as sex will be included in the model if they improve model fit without overfitting the data. If enough p-values fall below the Bonferroni-adjusted level to achieve no more than a 0.05 error rate, results may be reported with only adjusted p-values.
Sample size and power calculation
Sample size calculations were carried out based on expected differences within inflammatory monocytes (delta = 4.4, SD = 3.7), our primary variable. Values related to variability were suggested by previous studies on these same outcomes(2, 10).
To ensure equal distribution of CRE or RE bout first, eight independently generated randomization schedules were generated by the project biostatistician for the four groups (young physically active, young physically inactive, older physically active, older physically inactive, each stratified by sex). The biostatistician is not involved with recruitment or enrollment, and has no contact with the participants. A sequentially numbered list was kept, and as a participant is enrolled in the study and meets all study inclusion and exclusion criteria they are given the exercise bout assignment in order on the list. The co-investigator who consented the participant will assign the intervention, and there is no blinding.
Data storage and monitoring
Data entry will be performed by a member of the research team. A second member of the team will spot-check the data entry. Range checks will be conducted on every variable prior to statistical analyses. Biochemical assays will be performed in duplicate. All data is referenced by participant code. Data is stored on a password-protected university computer and server. Hard copies of data are stored in a locked cabinet in the Laboratory of Integrated Physiology. The informed consent is the only document with the participant’s name, and is stored in a separate cabinet. De-identified datasets may be uploaded to public databases and/or available by request to the PI. Scientific manuscripts will be used to disseminate the study results. Participants may request a copy of their DEXA results for personal informational use.
A data monitoring committee was not formed for this study. The exercise interventions in this protocol are well-established to be safe and recommended (“standard of care”) for the study population. The novelty of the protocol is in the study outcome variables, not the intervention. Any adverse event will be reported to the Institutional Review Board at the University of Houston. The investigators do not plan any formal audit, but the sponsor may randomly audit the study.