The study followed a double-blind crossover repeated measures controlled design. Environmental conditions were maintained the same in all testing sessions (Mean ± SD: 20 ± 1 °C, 775.6 ± 12 mmHg and 51.1 ± 6.1 %) for air temperature, barometric pressure and relative humidity respectively. The University of Greenwich granted institutional ethical approval, and all experimental procedures were carried out in accordance with the ethical guidelines of the World Medical Association Declaration of Helsinki. All study participants provided their informed written consent based on university-approved documents after the purpose of the research and experimental procedures were explained.
The participants were twelve healthy recreationally active adults, five females and seven males [Mean ± SD: age = 24 ± 3.8 y, height = 174 ± 9.1 cm, body mass = 69 ± 17.0 kg, body mass index (BMI; in kg.m−2) = 22.5 ± 3.85, body fat percentage (Fat%) = 21.3 ± 9.6 %, systolic blood pressure (SBP) =1 20.1 ± 9.7 mmHg, diastolic blood pressure (DBP) = 71.8 ± 5.7 mmHg]. All participants were screened prior to the start of the testing in order to determine that they were free from illness and any type of orthopedic limitation or injury. Exclusion criteria were as follows: A) History of any cardiovascular or respiratory disease, hypertension, liver or kidney disease, musculoskeletal or neuromuscular or neurological disease, autoimmune disease, cancer, peptic ulcers or anemia. B) Taking medications (except contraceptive pills), including those for heart, pulmonary, thyroid, anti-hyperlipidemic, hypoglycemic, anti-hypertensive, endocrinologic, psychotropic, neuromuscular, neurological, or androgenic conditions, as well as a family history of heart problems, high blood pressure, and/or stroke, and being pregnant or breastfeeding. C) Consuming any ergogenic aid or above habitual caffeine consumption rate (200 mg.day−1) for at least 6 weeks prior to the study, based on all types of caffeinated beverages (coffee, energy drinks, soft drinks, caffeine supplements or medications). All Participants refrained from taking any supplements for the duration of the study and were instructed to refrain from strenuous exercise or alcohol and caffeine consumption for at least 24 h before each test. Participants have also completed a 3-day 24-hr food diary with details about serving amounts for breakfast, lunch, dinner, snacks and additional meals.
Experimental procedures and protocols
All participants reported to the Physiology Laboratory on three separate occasions followed by 3 h fasting state in the first session, and 12 h overnight fast in the second and third sessions. Each testing session (between 8:30 and 11:30 am) was separated by at least three days within two weeks period. Session one was aimed to assess participant’s body composition and endurance performance relisted variables including peak oxygen uptake \( \left(\overset{\cdotp }{\mathrm{V}},{\mathrm{O}}_{2\mathrm{peak}}\right) \) and the intensity associated with the maximum fat utilization (Fatmax). During the second and third visits, after assessment of body composition, participants were randomized to ingest 1.5 g (3 × capsules) of either a multi-ingredient supplement (SHRED), (Shred-Matrix®, Muscle-Pharm Corporation, USA), or a placebo (PL) containing maltodextrin and hemp protein powder, presented in similar-appearance capsules to SHRED. Three capsules with similar coatings of either SHRED or PL were placed within an empty water cup and taken in the same way with a 240 ml of water. The SHRED capsules contained Green Tea Extract, Yerba Maté, Guarana Seed Extract, Anhydrous caffeine, Saw palmetto, Fo-Ti, Eleuthero root, Cayenne Pepper, and Yohimbine HCI. The thermogenic ingredients per capsule included approximately 70 mg of green tea leaf, 50 mg caffeine anhydrase, and 100 mg of Guarana seed extract. The exact content and other ingredients are in a proprietary blend.
Immediately following the anthropometric and body composition assessments, and ingestion, participants rested for 150 min in a semi recumbent position in quiet laboratory condition. Resting measurements involved heart rate (HR), blood pressure BP [systolic (SBP) and diastolic (DBP)] and RMR. Additionally, mood state was assessed just after the ingestion (150 min before exercise), immediately pre and post exercise. Perception of hunger was assessed every 30 min after the ingestion, immediately pre and post exercise.
For the estimation of FAO and CHO at rest and during exercise, breath by breath cardiorespiratory measurements included oxygen uptake \( \left(\overset{\cdotp }{\mathrm{V}},{\mathrm{O}}_2\right) \), carbon dioxide production \( \left(\overset{\cdotp }{\mathrm{V}}\mathrm{C},{\mathrm{O}}_2\right) \) and respiratory exchange ratio (RER), using an online gas analyzer (Metalyzer Cortex 3B, Leipzig, Germany), which was fully calibrated as described in previous similar studies [23].
Anthropometric and body composition assessments
Body mass (BM) and height were assessed, on a standard scale and stadiometer. Body composition was assessed for body fat percentage (%BF) by whole-body air displacement plethysmography (Bod Pod®, Life Measurements, Concord, CA), in accordance with the manufacturer’s instructions as detailed elsewhere [25]. Participants were tested wearing only tight fitting clothing (swimsuit or undergarments) and an acrylic swim cap. Thoracic gas volume was estimated for all subjects using a predictive equation integral to the Bod Pod® software. The calculated value for body density was used in the Siri equation [26] to estimate body composition. The complete body composition measurement was performed twice. If the %BF was within 0.05 %, the two tests were averaged. If the two tests were not within the 0.05 % agreement, a 3rd test was performed and, then, the average of three complete trials was used for all body composition variables.
Exercise protocol
All participants followed a ramp exercise cycling protocol using an electromagnetically braked cycling ergometer (Schoberer Rad Messtechnik, SRM, Ergo, Julich, Germany) during their first visit. The test was initiated with a power output of 30 W in females and 50 W in males and was increased in a ramp fashion, with the cadence maintained at 65–70 rpm throughout the whole test, until reaching volitional exhaustion defined as 1) meeting the at least two of \( \left(\overset{\cdotp }{\mathrm{V}},{\mathrm{O}}_{2\mathrm{peak}}\right) \) termination criteria: RER value > 1.1, heart rate within ten beats.min−1 of age-predicted maximum heart-rate, or achieving leveling-off of \( \left(\overset{\cdotp }{\mathrm{V}},{\mathrm{O}}_2\right) \), or 2) the participant could no longer maintain the required cadence for over 15 s despite verbal encouragement. Similar verbal encouragement was provided to all participants throughout the exercise tests. The cycling ergometer was calibrated before use. The cycling positions that were taken in the first test were re-applied in the following visits.
The following two visits involved the participants to follow a 30 min exercise cycling test at their individually determined Fatmax intensity. Additionally, the heart rate (HR) was measured continuously (Polar Sporttester, Polar Electro, Finland) and the rate of perceived exertion (RPE) using the Borg scale (6–20) was measured every 3 min during all tests.
Data processing and statistical analysis
The metabolic data of FAO and CHO were estimated using the stoichiometric indirect calorimetry equations (Eqs. 1 and 2), assuming minimal protein contribution during exercise.
$$ \mathrm{F}\mathrm{A}\mathrm{O} = 1.695 \times \kern0.5em \overset{\cdotp }{V}{O}_2\hbox{--} 1.701\kern0.5em \times \overset{\cdotp }{V}C{O}_2 $$
(1)
$$ \mathrm{C}\mathrm{H}\mathrm{O} = 4.585 \times \kern1em \overset{\cdotp }{V}C{O}_2\kern0.5em \hbox{--} \kern0.5em 3.226 \times \kern0.5em \overset{\cdotp }{V}{O}_2 $$
(2)
The FAO during exercise was determined based on averaging the last twenty min of the 30 min. Fatmax (g.min−1) was determined from the ramp test as the highest amount of FAO averaged over 30s. Fatmax corresponding intensity was determined as the power output (W) and relative intensity (%) relative to peak power (Ppeak), at which each participant achieved Fatmax [27].
Profile of mood state (POMS) questionnaire was analyzed for total mood disturbance total score, calculated for each participant by adding scores for Tension, Depression, Anger, Fatigue and Confusion and subtracting your Vigor score [28]. Hunger scale (1–10 scores) was used to indicate a satiety score, with a score of one being starving and ten being stuffed.
All data were described as means and standard deviations. FAO, CHO oxidation, and POMS questionnaire, were analyzed with 2x3 repeated measures ANOVA, to compare repeated conditions (SHRED vs. PL) over time (first hour pre, second hour pre, and post-exercise). The same procedure was used with Hunger Scale considering six time points (pre, every 30 min during exercise, and post), with Heart Rate and RPE considering 11 time points (rest, and every 3 min), and RPE considering ten time points (every 3 min). Post-hoc calculations were adjusted with Bonferroni method. Generalized Eta squared (η
2G
) and Cohen’s d were calculated as standardized effect size measures. Significance level was set at 0.05.