Multi-ingredient pre-workout supplementation changes energy system contribution and improves performance during high-intensity intermittent exercise in physically active individuals: a double-blind and placebo controlled study | Journal of the International Society of Sports Nutrition

The athletic gut microbiota | Journal of the International Society of Sports Nutrition


Study design

This study used a randomized, double-blind, crossover design with experimental trials conducted in the morning (7 to 10 AM) and each visit separated by 72 h. During the first visit, anthropometric measurements and the incremental running test was performed to determine V̇O2max and MAS. The following two visits, participants randomly consumed the MIPS or Placebo capsules one hour before performing a HIIE session. The V̇O2 and blood lactate concentration were monitored during the HIIE sessions to determine metabolic effort.


Twelve physically-active and healthy men were recruited for this study. The interview considered the following criteria of inclusion: being male, between 18 to 35 years age and practice aerobic exercise for at least 6 months. The participants were also excluded if they presented any medical contraindications that might interfere with their performance on the exercise protocol. All participants signed a consent form and were informed about the purpose of the study and the possible risks involved. This study was conducted after approval by the Ethics Research Group of the Federal University of Piauí, Teresina-PI, Brazil (Protocol number: 3.169.545) and according to the 2008 Revision of the Declaration of Helsinki [15]. The data collection process (CONSORT diagram) is shown in Fig. 1.

Fig. 1

Dietary intake assessment, supplementation protocol and anthropometric measurement

Participants were instructed to consume the same breakfast and to record food and fluid intake 24 h before each HIIE session. Participants were also instructed to avoid coffee, tea, alcoholic beverages, or any other stimulants, supplement or ergogenic substance during the study period, and were instructed to maintain their regular food intake. The consumption of kilocalories and macronutrients was calculated based on the Brazilian food composition table (TACO) to ensure that intake was similar in both experimental trials.

The MIPS used in this study was a commercially available product supplied by the manufactuer (Supley Laboratório, Matão, São Paulo, Brazil). The ingredients are listed in Table 1. The Placebo mixture (maltodextrin + juice Clight® containing maltodextrin, dyes, acidulants and flavorings, energy value = 34 Kcal, carbohydrates = 8.5 g) was diluted in 250 ml of water. Both placebo and MIPS mixture were identical in color and flavor. The MIPS or Placebo were delivered to the volunteers by a member of the laboratory who did not belong to the investigating research team to ensure a double-blind design. Participants ingested the substance in the laboratory one hour before the start of the experimental HIIE session. Body weight was measured using an electronic scale (Filizola PL50, Filizola Ltda., Brazil). A fixed stadiometer was used to measure height with an accuracy of 0.1 cm.

Table 1 Supplement ingredients

Incremental running test

Participants performed a maximal incremental test on a treadmill (Inbramed MASTER CI, Inbrasport®, Porto Alegre, Brazil) in an environment with controlled temperature and humidity. Expired gases were collected breath-by-breath with a silicon mask connected to the gas analyzer (Quark PFT – Cosmed®, Rome, Italy). The maximal incremental test was used to determine MAS for the prescription of HIIE sessions and V̇O2max for the characterization of the sample. Participants performed a warm-up of 5 min walking at 5 km/h before test. The initial test speed was set at 6 km/h and increased by 1 km/h every 2 min. Treadmill inclination was maintained a 1% and the test was terminated when the participant reached voluntary exhaustion. Verbal encouragements were provided to ensure that every volunteer ran to exhaustion. The MAS was assumed as the final velocity of the test. When the participants failed to complete a stage, the speed was selected according to the following formula: MAS = complete final stage velocity + [(time, in seconds, remaining in incomplete final stage / 120 s) * 1 km.h] [16]. The rate of perceived exertion (RPE) and heart rate (HR) were measured during the test using the 6–20 point Borg scale [17] and Polar S810i (Polar®, Finland), respectively.

High-intensity intermittent exercise (HIIE) protocol

For each trial session, participants performed a warm-up at 50% of MAS for 5 min. The HIIE involved running bouts of 15 s on the treadmill at 120% of the MAS, interspersed with 15 s of passive recovery. The inclination of the treadmill was maintained at 1% and the HIIE session was terminated when the volunteers achieved voluntary exhaustion. The total of sprint intervals performed and time to exhaustion under each condition were recorded.

Blood lactate

Twenty-five microliters of blood was collected from the volunteer’s right ear lobe at rest, immediately post, and 3, 5 and 7 min post-exercise. Lactate concentrations were obtained using the Yellow Spring 1.500 Sport lactate analyzer (Yellow Springs, USA). The delta lactate (highest value minus rest values – [∆La]) was utilized to compare conditions.

Oxygen uptake

V̇O2 was measured at rest, during the HIIE sessions and for 20 min after the end of the sessions. For data treatment, the mean V̇O2 during the HIIE session was analyzed for 15 s in every effort and every for pause. The T90% V̇O2max was determined from the average obtained in the 5-s periods due to the rapid changes in V̇O2 over time between sprint and pause periods in order to obtain greater sensitivity, and compared with the relative V̇O2max values. EPOC was obtained by subtraction of V̇O2 from rest of the mean V̇O2 of recovery [14].

Energy expenditure

The energy expenditure during the MIPS and Placebo trials was estimated from the contribution of the oxidative, glycolytic, and ATP-PC energy systems [18, 19]. The contribution of the oxidative energy system was estimated by subtraction of the resting V̇O2 from the mean V̇O2 calculated over the complete HIIE. For glycolytic energy system contribution, the Δ [La] was converted into oxygen equivalents assuming that the accumulation of 1 mmol/L of lactate is equivalent to 3 mLO2/kg body weight [19]. Finally, the sum of V̇O2-time average during the HIIE recovery periods (ΣEPOC) subtracted by resting V̇O2 was assumed for the contribution of ATP-CP [20,21,22]. In addition, the fast component of EPOC (i.e., estimated using V̇O2 kinetics as the product of V̇O2 amplitude and time constant using a bi-exponential fit) was calculated for the last effort utilizing the software Origin version 2019 (OriginLab Corporation, Microcal, Massachusetts, USA) and added to ΣEPOC. The oxygen equivalents were converted into energy equivalents considering 20.92 kJ for each 1 L of O2 used [23].

Statistical analysis

A power analysis (Power 1-β = 0.87) was performed a priori in G*Power software and indicated that effects could be detected with twelve participants. Data were reported as mean and standard deviation (SD). Data normality was verified using the Shapiro–Wilk test. The number of efforts performed, time to exhaustion, time above 90% V̇O2max, EPOC, dietary intake and oxidative, glycolytic and ATP-CP system contributions during MIPS and Placebo were analyzed by a paired t-test. For the lactate, a two way analysis of variance [group (MIPS versus Placebo) × time point (pre versus post)] was conducted followed by the Tukey’s post hoc test. Statistical significance was set at p <  0.05. The data were analyzed using SPSS version 22.0 (SPSS Inc., Chicago, IL).


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