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Participants
The 11 subjects in this study were all undergraduate students majoring in Physical Education from the Capital Institute of Physical Education and Sports in Beijing, China. Their mean age was 21.36 ± 2.46 years, height was 179 ± 5 cm, weight was 72. 8 ± 10.7 kg, percentage of body fat was 18.0 ± 6.7%, and body mass index was 23 ± 3 kg/m2 (Table 1). The inclusion criteria for participants were as follows: (1) They had to be within the age range of 18–28 years old and engage in at least two systematic training sessions per week. Participants were prohibited from using any additional dietary supplementation. (2) They were not allowed to consume androgens or other performance-enhancing drugs. Before the start of the experiment, all subjects were familiar with the experimental procedure and were required to sign an informed consent form. The minimal sample size of 12 (six in each group) was determined by a priori analyses using G*Power software (version 3.1.9.2) based on the following parameters: an alpha level of 0.05, a power (1-beta) equal to 0.8, and an effect size of 0.4.
Experimental design
A randomized crossover design was used in this study. Each participant performed three experiments. First, participants performed anthropometric measurements maximal incremental cycling test to determine maximal oxygen consumption (V̇O2), maximal heart rate (HRmax), and peak output power (PPO). They were then familiarized with the experimental procedures and squatting movement standards.
To assess arterial stiffness, we utilized the cardio-ankle vascular index (CAVI), which is an index that measures the stiffness of the arterial tree from the aorta to the ankle [22]. Participants randomly performed C-HIIT and R-HIIT respectively in the second or third experiments and were measured CAVI, systolic blood pressure (SBP), HRV and collected blood samples at previous, immediately, and 30 min after each exercise bout (Fig. 1). All visit sessions are separated by at least a week washout period between two HIIT sessions. Both C-HIIT and R-HIIT consisted of a 10-minute warm-up, 19 minutes of exercise intervention, and 30 minutes of sitting after exercise. All participants abstained from vigorous activity and from alcohol or caffeine intake over the day before the protocols. Participants completed the tests and exercise sessions from 9:00 a.m. to 12:00 a.m. of the day, and measurements were performed in a temperature-controlled (22–24 °C) and humidity-controlled (40–50%) room. Ethical approval was granted by the Capital University of Physical Education and Sports Ethical Committee. This study adhered to CONSORT guidelines for randomized controlled trials and had been registered on Chinese Clinical Trial Registry (www.chictr.org.cn, registration number: ChiCTR2200056897).
Exercise protocol of the present study
C-HIIT: cycling-type high intensity interval training; R-HIIT: resistance-type high intensity interval training
We used a HIIT protocol (10 × 60s high-intensity intervals separated by 60s of active recovery), which has been shown to be efficacious and practical in healthy young men [23]. During the second and third visits to the laboratory, participants were randomly assigned to one of the two modes of exercise: C-HIIT and R-HIIT (Fig. 1). Throughout the test, subjects received verbal encouragement. Our pilot study revealed that participants could perform two modes of HIIT with similar RPE. Therefore, the intensity of R-HIIT and C-HIIT was considered to be the same. This method has been used by other researchers [17, 18].
Participants in this study underwent a 10-minute warm-up session before engaging in the C-HIIT training program, including dynamic stretches and 3 minutes of cycling at 50 W. Following a 1-minute transition period, the official test began. The C-HIIT program, conducted using a cycle ergometer (ergoline 100 K, Germany), consisted of 10 60-second working intervals at 90% PPO, with each interval followed by a 60-second active recovery period at 25% PPO. Throughout the exercise, participants were instructed to maintain a pedal cadence between 60 and 65 rpm.
Before starting R-HIIT, participants performed approximately 10 minutes of warm-up, including dynamic stretches and squats without load. The R-HIIT protocol consisted of 10 60s working intervals, each separated by 60s recovery periods. During the working intervals, participants performed barbell back squats with a load of 20% bodyweight. To ensure proper form, participants placed the barbell on the trapezius and held it with both hands with the assistance of the researcher. They were instructed to squat down to approximately 90° knee flexion, maintaining contact between their feet and the floor, and were supposed to achieve full hip and knee extension at the end of each repetition. The eccentric phase of each squat lasted 1 second, while the concentric phase was performed as fast as possible to a standing position. The maximum number of squats completed per working interval was 30 reps. To control the tempo of the movements, a metronome set at 60 beats per minute was utilized. If participants were unable to complete the movement at the prescribed tempo, a brief rest in a standing position with the load was allowed. After each working interval, the researcher removed the barbell and repositioned it on the participant’s trapezius 5 seconds before the next exercise bout.
Experimental measures
Anthropometric measurements
All anthropometric data are shown in Table 1. Participants’ height (cm) and body weight (kg) were measured using an ultrasonic tester (DHM-200, China). BMI was then calculated by dividing the subjects’ weight (kilograms) by the square of their height (meters). Additionally, body composition was assessed by dual X-ray absorptiometry (Lunar, United States).
Incremental exercise test
V̇O2max is determined through a graded incremental exercise test (GXT) conducted on a cycling ergometer (ergoline 100 K, Germany). The test started with a 5-minute warm-up period at 50 W power. Subsequently, the workload was increased by 15 W every minute from 70 W until the participants reached voluntary exhaustion. Exhaustion was defined as the inability to maintain a speed of 60 revolutions/min for more than 5 seconds in succession despite verbal encouragement. During the GXT, expired gases were analyzed using a gas analyzer (AEI moxus, United States). Additionally, heart rate (HR) was continuously measured through the Polar RS400 to determine if participants reached their V̇O2max, they had to meet at least three out of the following four criteria: (1) a plateau of V̇O2 (i.e., change ≤2.1 mL/kg/min), (2) a final respiratory exchange ratio (RER) of ≥1.1, (3) maximum heart rate within 10 bpm of the age-predicted maximum [210-(0.65 × age)] [18, 24], and (4) a rating of perceived exertion (RPE) of ≥18. V̇O2max, calculated as the mean of the highest values obtained during three consecutive 10-second periods (30 seconds total), is presented in Table 1.
When the exercise intensity could not be maintained for the full 60 seconds, PPO was identified at the work rate coincident with volitional fatigue. PPO was calculated as Wfinal + (t/T)•Winc, Wfinal is the last power output (W) completed for 60 seconds, t (s) is the amount of time reached in the final uncompleted stage, T (s) is the duration of each stage (60 s), and Winc (W) is the workload increment (15 W).
Cardio-ankle vascular index and blood pressure text
Before warm-up, immediately and 30 min after both C-HIIT and R-HIIT, CAVI was measured using the VaSera VS-1500 vascular screening system (Fukuda Denshi, Beijing, China). Subjects were lying supine on a bed with all four limbs extended, and their information, such as name, gender, birth date, height, and weight was entered into the machine. The heart sound detector was placed near the sternum to monitor the heart sounds, the electrodes were clamped on the right and left wrists, and four cuffs were wrapped around the right and left upper arms and ankles [25]. When the first and second heart sounds were detected in the phonocardiogram, the START button was pressed. The average of the right and left CAVI (⊿CAVI) was calculated, and its change from baseline in the same trial was used for later analysis. The VaSera VS-1500 vascular screening system also measured blood pressure (BP).
Heart rate variability
HRV was measured simultaneously with ⊿CAVI. The assessment of all variables related to HRV was conducted at three different time points: (a) before warm-up, (b) immediately after exercise, and (c) 30 minutes. Cardiac autonomic modulation in HRV was measured using an electrocardiographic workstation (Shenzhen Boying Medical Instrument Technology Co, China) with subjects in the supine position and electrocardiogram electrodes placed on their wrists. The system obtained the values of every R-R interval independently, and 5 minutes were chosen for analysis. The Kubios software (v.3.5.0, HRV analysis, Finland) was used for HRV data processing. In the present analysis, the time domain variable of HRV utilized was the root mean square of successive differences between adjacent normal R-R intervals (RMSSD). The frequency domain variable was the high-frequency (HF) power of 0.15–0.4 Hz, as well as the LF/HF ratio. Among these parameters, the RMSSD and HF represent vagal activity, and LF/HF ratio is a measure of the cardiovascular autonomic balance. To address the skewness and violation of normality assumptions in the raw values of RMSSD, HF, a natural log (ln) transformation was applied (lnRMSSD, lnHF).
Blood sample collection and analysis
The whole blood (15 ml) was drawn from an antecubital vein by a skilled nurse previous, 5 minutes and 35 minutes after each exercise bout. After drawing the blood samples, the whole blood samples were allowed to clot at room temperature for 30 min, and then centrifuged at 3000 rpm for 10 min at 4 °C. Following centrifugation, the separated serum samples were frozen and stored at − 80 °C. The serum levels of cTnT and NT-proBNP were measured using ELISA kits (Albion, China). The kits had an intra-assay coefficient of variation of less than 10%. All analyses were conducted using an automatic microplate reader (E 601, Germany).
Statistical analyses
Statistical analyses were performed using SPSS Statistics (version 26, IBM, United States). Data were presented as mean ± standard deviation (SD). Normality assumptions were checked using Shapiro-Wilk tests. A 2 × 3 two-way ANOVA with repeated measures was used to examine the changes in ⊿CAVI, SBP, lnHF, lnRMSSD, LF/HF ratio, cTnT and NT-proBNP across the two modes (C-HIIT and R-HIIT) observed points (Pre, 0-min post and 30-min point). Post-hoc analyses, using the Bonferroni’s correction factor were performed for cases in which the main effect was significant. Partial eta squared (ηp2) was calculated to determine the magnitude of main and interaction effects and was categorized as small (0.01), medium (0.06), and large (0,14), respectively. Cohen’s d was used as a measure of effect size, with small, medium, and large effects equal to 0.2, 0.5, and 0.8, respectively. All analyses used an alpha level of P < 0.05 to indicate statistical significance, while a highly significant difference was indicated by P < 0.01.
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