Forty-five sedentary men were recruited for this study (Characteristics are represented in Table 1). Inclusion criteria to be included in the study were: 1) had not participated in regular physical activity in the last six months, 2) not meet the recommendations of the American College of Sport Medicine (150 min moderate-intensity exercise or 75 min vagarious exercise per week), 3) being non-smokers, 4) no history of chronic diseases particularly cardiovascular disease, and 5) were not taking medication in the last six months. Subjects with joint or skeletal muscle disorders were excluded from the study. Subjects were also screened for normal resting blood pressure (BP) and resting HR (RHR) by an experienced clinical exercise physiologist. Each of the subjects completed an informed consent form describing experimental procedures, risk, and potential benefits associated with taking part in the study before the beginning of the study. The study procedures were reviewed and approved by the local University Research and Ethics Committee (Ethics code: IR-IAU1398-18). All procedures were performed according to the latest revision of the Declaration of Helsinki.
Subjects were randomly allocated into three groups: High-volume moderate-intensity training (HVMIT; n=15), Low-volume high-intensity training (LVHIT; n =15), or control (n = 15) (Figure 1). Study measurements (Resting HRV, PWV, resting blood pressure, body composition, and VO2max) were taken 48 hours before beginning the training intervention and 48 hours after the last session of both training protocols. Subjects were asked to refrain from any caffeine-contained beverages and moderate to vigorous physical activity 24 hours before measurements. All measurements were conducted in the morning between 8 a.m. to 10 a.m. under the same environmental conditions (~20°C and ~55% relative air humidity).
Heart rate variability
After 12 hours of fasting, the subjects visited the laboratory in the morning (8:00 am to 10:00 am). Subjects were seated in a quiet room with the light turned off (22–23°C) before conducting the evaluations. The RR recordings were captured by a Heart Rate Monitor (Polar Electro, Kempele, Finland). The HRV test was conducted in a supine position for 25 minutes. All the procedures were performed according to the standards proposed by the Task Force of the European Society of Cardiology . The resting HRV was evaluated for 25 minutes in the supine position with the first 5 minutes devoted to signal stabilization and not included in analyses. All artifact and ectopic data were filtered and corrected with a minimum protection zone of six beats. All recording data with more than 5% error were not included. A computer software program (The Biomedical Signal and Medical Imaging Analysis Group, Department of Applied Physics, University of Kuopio, Finland) was used to analyze both time and frequency domain data . The time-domain of HRV included: the Standard Deviation of RR intervals (SDNN) and index of overall variability, and the Root Mean Square of Successive Differences between RR intervals (RMSSD) which represents vagal modulations. The frequency-domain parameters included the low-frequency (LF: 0.04–0.15 Hz) and high-frequency bands (HF: 0.15–0.40 Hz) were calculated by using fast Fourier transformation (FFT; welch, 256 points Hanning-windowing, Kubios HRV Analysis, Biosignal Analysis, and Medical Imaging Group, University of Eastern Finland, Finland) for analysis of power spectrum. The ratio of the LF to HF (LF/HF) bands was also evaluated.
Resting Blood Pressure
Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured with an electronic sphygmomanometer (BPM AM 300P CE, Kenz, Suzuken Company, Japan) before HRV measurement and according to the guidelines of the European Society of Hypertension . Before the measurement of blood pressure, subjects were seated on a comfortable chair for 10 minutes.
Pulse Wave Velocity
PWV (Arterial stiffness index) was measured with a Vascular Explorer (Enverdis, Jena, Germany). Measurement was taken after 10 min of rest in a supine position. Analysis of PWV and blood pressure were performed by photoplethysmographic sensors and inflatable upper arm and lower leg cuffs techniques, as described elsewhere .
The bio-impedance analyzer (Medigate Company Inc., Dan-dong Gunpo, Korea) was used to measure fat-free mass (FFM) and body fat percentage. Body mass (kg) was divided by the square of the height (m2) for BMI calculation.
The maximal oxygen uptake (VO2max) test was initiated with a speed of 6 km∙h-1 on a motorized treadmill (H/P/Cosmos, Pulsar med 3p- Sports & Medical, Nussdorf-Traunstein Germany). The treadmill speed was increased by 1 km∙h-1 every 3 minutes until subjects were physically exhausted and unable to continue the test. The treadmill grade was set at 0.5 degrees throughout the test. A gas analyzer system (Metalyzer 3B analyzer, Cortex: biophysik, GMbH, Germany) was used to measure oxygen uptake (VO2) and was calibrated before each test based on the manufacturer's instructions. The highest 60-s VO2 value during the test was considered the VO2max. The last completed 3-min velocity of the treadmill test was identified as the maximal testing velocity (Vmax). The details of the VO2max test were previously described elsewhere .
Nutrient intake and dietary analysis
Subjects were instructed to document all food intake as precisely as possible for three days (two weekdays and one day on the weekend) throughout the study period. Total energy (kcals per day) and intake of carbohydrates, fats, and proteins (grams per day) were calculated. All subjects’ dietary logs were assessed using Diet Analysis Plus, version 10 (Cengage, Boston, MA, USA).
Running Training protocols
The running training program for the HVMIT protocol included: first and second weeks at 50% of VO2max during 45 min, third to seventh weeks at 55% of VO2max during 50 min, and the last five weeks at 60% of VO2max during 60 min. The LVHIT protocol was as follows: first two weeks at 70% of VO2max during 25min, third to seventh weeks at 80% of VO2max during 35min, and in the last five weeks at 85% of VO2max during 40min. Caloric expenditure for both training protocols was equated (14 kcal/kg of body mass per week), and the amount of distance ran per week was similar between protocols (17.5 km). Both protocols were performed on a motorized treadmill (H/P/Cosmos, Pulsar med 3p- Sports & Medical, Nussdorf-Traunstein Germany). The intensity of exercise in both protocols was controlled and initially set by Vmax. In addition, during all exercise sessions, subjects used an HR monitor (Polar V800, Polar Electro Oy, Finland) to monitor their HR. Moreover, we calculated the actual amount of regular exercise completed according to the subjects' compliance. All subjects' adherence calculation was conducted by dividing the number of minutes completed within the identified HR range each week by the minutes prescribed (Table 2). The VO2max test was conducted at weeks 4 and 8 to maintain the target intensity. Both training groups performed three supervised exercise sessions per week for 12 weeks. The exercise training protocols details are shown in Table 2. The control group was asked to maintain their daily routines and refrain from any additional physical activity during the study.
The normality of all data was evaluated with the Shapiro-Wilk test. A one-way ANOVA was applied to compare all baseline data among three groups to ensure no significant differences between groups. A two-way ANOVA with repeated measures (Groups * time) was used to compare three groups' changes for all variables. If group x time interactions turned out to be significant, a Bonferroni post hoc test was calculated. Additionally, effect sizes (ES) were determined from ANOVA output by partial eta-squared. Moreover, within-group ES were computed using the following equation: ES= (mean post-mean pre)/SD . In accordance with Hopkins et al. (2009) ES were considered trivial (< 0.2), small (0.2-0.6), moderate (0.6-1.2), large (1.2-2.0) and very large (2.0-4.0). Data were described by means and SD (±). The level of significance was set at p˂0.05. All statistical analyses were computed using SPSS for Windows, version 23.0 (SPSS Inc., Chicago).