Participants
A total of 13 men and 2 women were actively recruited for the study. All participants were evaluated for safe exercise participation using an American College of Sports Medicine (ACSM) risk factor assessment and informed of the purposes of the study before signing an informed consent document approved by the Virginia Commonwealth University (VCU) Institutional Review Board. Inclusion criteria included men and women between the ages of 18–35 years old who were minimally active—at or below 3 × 30 min of activity/week, and had a body fat ≤25 % for men and 32 % for women. Exclusion criteria were any person exceeding the body fat cut off, orthopedic limitations preventing full participation in the study, pre-diabetes or diabetes mellitus, reported hypothyroidism, renal disease, and/or anyone considered high risk for exercise participation based on current ACSM clinical guidelines.
Experimental Protocol
All experimental procedures were performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the VCU Institutional Review Board. The design (Fig. 1) was similar to previous SIT studies and consisted of a 1-week intra-subject control period. During this period, participants performed baseline (B) and pre-training (PRE) oral glucose tolerance tests (OGTTs). Participants then completed two different acute SIT protocols—Tabata and Wingate, utilizing a counter balanced cross-over trial design, with each training bout separated by no less than 1 week. Subject 1 was randomly assigned to either Tabata or Wingate first, completing the other session second; each subsequent subject was then assigned to the opposite group first. All exercise took place using a mechanically braked Monark Peak Bike (Monark Exercise AB, Sweden) equipped with an SRM power meter (SRM Service Center, Inc., Colorado Springs, CO). Blood samples (~10 ml) were taken immediately following, and 1 h after each training session, as well as 24 h after exercise; this corresponded to the initial resting sample prior to the post-exercise OGTT. Women participants were tested during the follicular phase of their self-reported menstrual cycle to minimize the impact on cytokine levels.
Dietary Control
Each subject was asked to complete a 3-day dietary recall form prior to completing any blood analysis. Dietary analysis was performed by a registered dietician for later comparison, and participants were asked to pick 1 day within the recall and repeat those meals the day before each OGTT, recording the meals for those days. Additionally, each participant completed all training sessions after a 12-h fast, including alcohol and caffeine, and they abstained from significant activity 24 h prior to all exercise sessions and OGTTs.
Preliminary Testing and Evaluation
On the initial assessment day (day 3) and during the OGTT, participants completed body composition analysis using bioelectrical impedance analysis (RJL Quantum IV, RJL Systems, Inc., Clinton Township, MI), where participants lay supine for a period of 20 min to allow body fluids to equilibrate across the body. During this time, small electrodes were placed on the right ankle and wrist. Body composition was then estimated using web-based software (RJL Interactive Online BIA, RJL Systems Inc., Clinton Township, MI). Following the initial OGTT, physiologic testing included bicycle VO2Peak testing. Testing was completed on the SRM equipped Monark bike; power (W) was measured for each stage. VO2 and HR were measured continuously using a Parvo OneMax system (Parvo Medics, Salt Lake City, UT) and Polar HR monitor (Polar Electro Inc., New Success, NY), respectively. Participants were instructed to pedal at their preferred cadence throughout testing. The initial workload was set at 1.5 KP with an approximate work rate of 100 W, increasing by 0.5 KP every 2 min until volitional exhaustion was reached, or the subject could not maintain their chosen cadence. Peak power output at VO2Peak was calculated as the highest average 1-min power output achieved during the final stage of testing; this power output was termed Power at VO
2Peak
.
Exercise Protocols
All SIT sessions began between 0700 and 0900, and each subject’s sessions took place at the same time of the morning. All sprint bouts began with a 10-min unloaded warm-up at ~70 rpm. Participants then pedaled against a resistance equivalent to 7 % (0.07 kg kg−1) body mass for Wingate sprints and slightly lower 5 % (0.05 kg kg−1) body mass for Tabata. The former resistance has been shown to produce optimal power output and reliable measurement [12], while the latter was shown to be optimal during pilot data work prior to the study. Participants were instructed to pedal as fast as possible for ~2 s before the load is applied and to continue to crank while being provided with vigorous verbal encouragement throughout each sprint. The Wingate protocol consisted of a total of five 30-s sprints with approximately 4-min recovery (i.e., very slow unloaded pedaling), while those completing the Tabata protocol consisted of ten 20-s sprints with 10-s recovery. The number of intervals performed for each protocol was chosen based on pilot data indicating a close match in total work (kJ).
Peak and mean power (W), as well as total work, were measured and stored using the SRM power meter and downloaded for later analysis using commercially available software (Training Peaks 3.0, Training Peaks, Boulder, CO). Blood lactate samples (5 μl) were measured from the fingertip using a small plastic lancet prior to exercise, immediately following, 1 and 3 min after exercise, and analyzed using a Lactate Scout Analyzer (EKF diagnostic sales GmbH, Barleben/Magdeburg). Each sprint session lasted between 15 and 30 min with warm-up. For descriptive purposes, the relative average power output during each SIT session was expressed a % Power at VO
2Peak
.
Blood Analysis
Hemoglobin concentration (g dL−1) and hematocrit (%), using the micro-hematocrit method, were measured in duplicate and then used to estimate percentage changes in PV [6]. An indwelling venous catheter was inserted to allow for convenient blood draws. Blood samples (~10 ml) were collected together using gray top sodium fluoride tubes (OGTT) and gold top serum-separator tubes (cytokines) throughout testing and then centrifuged after each session at 4000 rpm for 15 min at 4 °C. Separated plasma was immediately removed and stored in capped 1.5-ml polypropylene tubes frozen at −80 °C until later analysis.
Oral Glucose Tolerance Tests
OGTTs were completed following insertion of a catheter. Blood (~10 ml) was drawn before, as well as 30, 60, 90, and 120 min after ingestion of a 75 % Glucola drink (Fisher Science Inc., Philadelphia, PA). Plasma glucose concentrations (mg dl−1) were measured using the auto-analyzer glucose oxidase method, while plasma insulin concentrations (mU l−1) were determined by ELISA (R&D Systems, Inc, Minneapolis, MN). The coefficient of variation (CV) for baseline Cederholm Si was 4.8 %.
Inflammatory Markers
Inflammatory markers of interest included IL-6 (IL-6 B), IL-10 (IL-10 B), and TNF-α (TNF-α B) measured during baseline testing periods, as well as following each bout of SIT based on the time periods reported in prior research [22–24]. At baseline, samples were analyzed from the blood taken at minute 0. On SIT days, 10 ml of blood was taken prior to, immediately following exercise (P), and 1 h later (P 1). A final cytokine measurement was taken prior to the OGTT ~24 h after the SIT bout (P 24). Plasma concentrations of IL- 6, IL-10, and TNF-α were determined using interleukin-specific Humakine ELISA kits (R&D Systems, Minneapolis, MN), each completed according to manufacturer’s instructions. Coefficients of variation (CV) for IL-6, IL-10, and TNF-α were 9.9, 6.1, and 6.6 %, respectively.
Statistical Analysis
Data analysis was performed using commercially available software (Jump 13.0, SAS Institute Inc, Cary, NC). During the design process, power analyses run for cytokines and Si estimated that an N of 15 provides a power of 0.85. All data are presented as means ± SD. Area under the curve (AUC) was calculated using the trapezoidal rule, while the Cederholm index, which represents peripheral Si, was calculated using the formula:
$$ \frac{\mathrm{Cederholm}\ {\mathrm{S}}_{\mathrm{i}} = 75,000 + \left({G}_0-{G}_{120}\right) \times 1.15 \times 180 \times 0.19 \times \mathrm{B}\mathrm{W}/120 \times {G}_{\mathrm{mean}} \times \log\ \left({I}_{\mathrm{mean}}\right)}{1000} $$
BW is the body weight, G
0 and G
120 are plasma glucose concentration at 0 and 120 min (mmol l−1), and I
mean and G
mean are the mean insulin (mU l−1) and glucose (mmol l−1) concentrations during the OGTT.
All exercise responses (Si, glucose AUC, insulin AUC, and cytokines) are reported as absolute values and changes from baseline. Data were analyzed using absolute change responses from baseline ± 95 % confidence intervals; 95 % CI changes that failed to cross 0 (i.e., 0 change) were considered significant. Dependent t tests were run to compare change in Si following Tabata and Wingate SIT from baseline and at 24 h. IL-6, IL-10, and TNF-α were compared between SIT groups using a similar repeated measures ANOVA model, though with a four-level time indicator (baseline, post, 1 h, 24 h). Finally, Pearson’s correlation coefficients were calculated to examine the relationships between Si and changes in cytokine response.