Metabolic Health, IGF-1, and Cardiovascular Fitness Responses to High-intensity Interval Training and Moderate-intensity Continuous Training in Combination with Superimposed Neuromuscular Electrical Stimulation

Body composition via Bioelectrical Impedance Analysis (Tanita 418-MA, Japan)

时间窗: From enrollment to the end of treatment at 8 weeks

Weight (kg) Total body fat (kg) Total muscle mass (kg) The anthropometric parameters were assessed using Bioelectrical impedance analysis (Tanita 418- MA Japan) before all testing sessions.

Height (cm) via a stadiometer (Holtain Ltd., UK).

时间窗: From enrollment to the end of treatment at 8 weeks

Height was measured through a stadiometer in the standing position (Holtain Ltd., Crymych, UK).

Heart rate (bpm) via 12-lead ECG

时间窗: From enrollment to the end of treatment at 8 weeks

Heart rate was monitored and recorded throughout baseline and follow-up screenings and all training sessions using 12-lead ECG.

Blood Lactate Concentration (mmol/L) via Lactate Pro 2 analyzer

时间窗: From enrollment to the end of treatment at 8 weeks

In each GXT testing session (pre-post), blood samples were collected from the earlobe using a Lactate Pro 2 handheld analyzer (LT-1730, Arkray Inc, Kyoto, Japan) to determine blood lactate concentrations testing session (baseline) and at the end of every two minutes interval.

SERUM INSULIN-LIKE GROWTH FACTOR-1 (IGF-1 ng/mL)

时间窗: From enrollment to the end of treatment at 8 weeks

A 5 mL fasting blood sample was drawn from the brachial vein 30 minutes before baseline and follow-up IVO2max tests to measure serum IGF-1 concentrations. Participants rested for 15 minutes before sample collection. Samples were stored at -80 °C and analyzed post-study. Serum IGF-1 levels were measured using Elabscience ELISA kits (detection range: 1.56-100 ng/mL; sensitivity: 0.94 ng/mL) per the manufacturer's instructions.

VO2 - Volume of oxygen (ml/kg/min) VCO2 - Volume of carbon dioxide (ml/kg/min)

时间窗: From enrollment to the end of treatment at 8 weeks

During all sessions, VO2 and VCO2 were measured using indirect calorimetry (CareFusion MasterScreen CPX) on an Ergoline Ergoselect 100/200 cycle ergometer. Data was averaged over 15-second intervals during all baseline and follow-up screenings and all training sessions. VO2 and VCO2 were also used to calculate respiratory exchange ratio (RER) during all sessions dividing the VCO2 by VO2.

Energy expenditure (L/min) via indirect calorimetry (CareFusion MasterScreen CPX).

时间窗: From enrollment to the end of treatment at 8 weeks

During all sessions, energy expenditure was measured using indirect calorimetry (CareFusion MasterScreen CPX). Data was averaged over 15-second intervals, and substrate utilization was calculated using standard formulas. * (GOR)- Oxidation rate of sugar (g/min): 4.585 × VCO2 (L/min) -3.2256 × VO2 (L/min) * (GO)- Oxidation amount of sugar (g): Oxidation rate of sugar × Time (min) * (FOR)- Fat oxidation rate (g/min): 1.695 × VO2 (L/min) -1.701 × VCO2 (L/min) ④ (FO)- Oxidation amount of fat (g): Oxidation rate of fat × Time (min) * (EER)- Energy consumption rate (kcal/min): 3.716 × VO2 (L/min) +1.332 × VCO2 (L/min) ⑥ (EE)- Energy expenditure (kcal): \[3.716 × VO2 (L/min) +1.332 × VCO2 (L/min)\] × Time (min)

Neuromuscular Electrical Stimulation (NMES) Protocol

时间窗: From enrollment to the end of treatment at 8 weeks

The NMES protocol was administered via a four-channel COMPEX SP4.0 (Medicompex SA, Ecublens, Switzerland) electric muscle stimulator using biphasic symmetric rectangular pulsed currents set at 300 μs. COMPEX self-adhesive electrodes were used during muscle stimulation with the COMPEX device. Positive snap electrodes (5×5 cm) with a membrane depolarization that stimulate a 25 cm2 area of the muscle surface were placed on the proximal insertion of vastus medialis and vastus lateralis. The other negative electrode (10×5 cm), measuring 50 cm2 was placed over the femoral triangle, 1-3 cm below the inguinal ligament.

Low-frequency Neuromuscular Electrical Stimulation (NMES)

时间窗: From enrollment to the end of treatment at 8 weeks

The low-frequency NMES protocol was administered via a four-channel COMPEX SP4.0 (Medicompex SA, Ecublens, Switzerland) electric muscle stimulator using biphasic symmetric rectangular pulsed currents set at 200 μs. Low-frequency NMES protocol was performed with a duty cycle of 20 seconds on (stimulating) and 20 seconds off (no stimulation) and the pulse width was set at 300 μs (warm-up frequency: 3 Hz, training frequency: 20 Hz, wave: square waveform) to the quadriceps muscle throughout 24 sessions. The duration of training was 30 minutes for sessions 1-6, 36 minutes for sessions 7-12, 42 minutes for sessions 13-18, and 48 minutes for sessions 19-24.

High-frequency Neuromuscular Electrical Stimulation (NMES)

时间窗: From enrollment to the end of treatment at 8 weeks

High-frequency NMES protocol was administered via a four-channel COMPEX SP4.0 (Medicompex SA, Ecublens, Switzerland) electric muscle stimulator using biphasic symmetric rectangular pulsed currents set at 300 μs. High-freqeuncy NMES protocol consisted of a 5-minute warm-up (65% VO2max) followed by a 1-minute exercise at 120% VO2max and then a 1-minute "loadless" cycling. This interval was repeated 8 times in sessions 1-6 and progressed to 14 repeated intervals by the 24th session. Participants also received an additional NMES treatment with a duty cycle of 12 seconds on (stimulating) and 8 seconds off (no stimulation) and a pulse width of 300 μs (training frequency: 45-60 Hz, wave: square waveform) to the quadriceps muscle throughout 24 sessions.