Subjects

A sample of 9 or 13 subjects was calculated in order to find differences in cognitive or physical tests. This was done by using PRIMER software 3.02 (PRIMER-E Ltd, United Kingdom), after establishing a confidence level of 95% and a power of 80%. Nonetheless, 14 volunteered male soldiers, members of the National Army of Colombia Seventh Division, were included in the study. No cigarette smokers or users of psychoactive substances, or subjects under any medication (e.g. beta blockers) were included. People who consumed more than 600íml of coffee or more than 5 units of colas per day were excluded [11].

All participants underwent a complete physical examination and an electrocardiogram to determine their physical and mental conditions and all were diagnosed as healthy. In addition, they all signed the informed consent before the physical and cognitive tests. The procedures performed were endorsed and approved by the Indeportes Antioquia Research Committee and the National Army Research Committee in order to provide protection to the study participants.

Experimental design

This research used a double-blind, placebo controlled, randomized, crossover design, in which each participant visited six times the Exercise Physiology Laboratory from Indeportes Antioquia, in Medellín, Colombia, at 1540ím above sea level. Three fitness tests and two cognitive condition tests were applied in each visit. All evaluation sessions were separated between 48 or 72íhours and three subjects were evaluated per day. In the first session the subjects performed the tests, without drink consumption, to become familiar with their development. Testing was always conducted at the same time (2ípm-5ípm) in an environment with controlled temperature and humidity of 25íC and 60%, respectively. A training specialist and a psychologist with extensive experience in sports fields were responsible for measuring all variables.

Participants maintained their regular food intake, workplaces and recreational activities during study participation, but did not train at high intensity eight hours before the tests, did not eat heavy meals two hours before the tests and avoided caffeine consumption during the day of evaluation. Since the participants were soldiers, they had a very controlled schedule regarding food and exercise every day. So we are sure that during the day of evaluation the variation in these variables was kept to the minimum. All subjects used appropriate clothing for physical exercise.

Before developing the tests all subjects were randomly assigned (with the help of Excel 2007, Microsoft Co, USA) to a treatment (a placebo drink, a caffeinated drink, a caffeine and taurine combined drink, a drink with only taurine, or a commercial energy drink) and after 45íminutes performed each of the tests. All subjects received all beverages during the study. The composition of the five beverages is illustrated in Tableí1.

Table 1 Beverages composition per serving (250íml) used in the study Full size table

The first four beverages were developed with a flavor profile similar to the commercial energy drink brand (Red Bullí). For the design and formulation of each treatment, final contents of other ingredients such as sweeteners, acidulants, stabilizers, and colorings were determined apart from the major bioactive components, to ensure a close level to the physicochemical properties such as acidity, pH, refractometric degrees, color, smell and sensory profile similar to the commercial energy drink.

Carbonated-based water was used for all drinks and the appropriate powder mixture, previously formulated, was added. All bioactive and additional ingredients were selected from certificated suppliers and were of food grade.

Brix (Mettler Toledo RE50 refractometer), pH and acidity (Mettler Toledo Seven Easy pH meter), and color (Hunter Lab D25LT colorimeter) were analyzed in all beverages.

In order to ensure similarity between all beverages, and keep the double-blind design of the study, organoleptic properties such as smell, color, sweetness, acidity, body and overall impression were evaluated. This analysis was supported by the Colcafí expert panel (Medellín, Colombia). Decisions on formulation adjustments were based on the results obtained and analyzed with FIZZ Sensory Analysis Software (France).

All treatment beverages, including the commercial one, were pasteurized at 80íC for 15íseconds, bottled in 250íml polyethylene terephthalate bottles, identified with numeric codes and administered in identical conditions to the participants. The day of the experiment, each participant consumed all 250íml of the assigned drink in a period of 10 to 15íminutes.

Evaluations

Food consumption

A 24-hour recall and an analysis of the complete menu provided by the Army to the soldiers were used to determine the dietary intake of the subjects in the study. Portion sizes, brands and cooking methods were recorded. We used the official food composition table from University of Antioquiaís School of Nutrition and Dietetics (Medellín, Colombia) to determine the calories and nutrients consumed by the tested subjects and the average values were used in the analysis of consumption.

Level of physical activity

The level of physical activity was measured using the Global Physical Activity Questionnaire (GPAQ) of the World Health Organization [12]. Subjects were interviewed to remember the number of days from the last week when they did vigorous intensity or moderate intensity activities in three aspects (work or home activities, travel to different places or leisure activities) and the number of hours and minutes per day of different activities performed. This multidomain questionnaire was used since it was validated in both developed and developing countries and several populations including Latino people [13]-[15]. It is now used worldwide and this fact enables comparisons across diverse populations.

Physical tests

Determination of cardiorespiratory fitness

All participants completed a set of ergospirometry tests that allowed us directly monitoring their cardiorespiratory behavior. An open circuit spirometry (Oxycon Delta, Jaeger, Spain) previously calibrated was used. A ramp protocol was used in test performing [16]. After explaining the test to each subject, the resting ergospirometry parameters were taken and the test was started with a three minutes warming up period at a speed of 3.5 mile/h with a band inclination of 1%, then the speed was increased by 0.5 mile/h every minute until exhaustion. To measure participant recovery, subjects continued walking with an intensity and duration similar to the warming up period for three minutes, followed by other two minutes of passive recovery in standing position. After the test each subject was disconnected from the computer and the information obtained was recorded. Maximum oxygen consumption (VO 2 max), maximum heart rate (HRmax) and time to exhaustion (TTE) were determined from test results.

Strength calculation

A static dynamometry test of the upper limbs was performed. We used handgrip strength, which has been considered a simple, fast and reliable test for measuring human strength in different populations [17]-[20]. For handgrip strength measurements (finger flexor force), a previously calibrated Takei Kiki Kogyo digital dynamometer (Japan), with a transducer 1270A, series 86002, 90íkg, was used. Each of the participants performed a ten minutes guided warming up, focusing on the muscle groups that were evaluated. Subsequently, the evaluated subject stood up and held the transducer, in such a way that the upper end of this rests on the palm, under thenar and hypothenar regions, and the lower end on the second to the fifth finger second phalanx. Then, with the upper limb away from the body and no support points for the transducer, the soldier proceeded to perform bilateral maximum handgrip strength for five seconds; this procedure was repeated after one minute of recovery and the highest value was recorded [21].

Power calculation

Three vertical jumps were performed on a contact, previously calibrated, platform (Newtest Power timer series 10327) that calculated the flight time. Data obtained allowed to calculate the height reached and the subject mechanical power. Functioning of the test was explained to participants; then, there was a warming up emphasizing the muscles of the lower limbs. After subjects performed three nonconsecutive free jumps using arms, the height was measured by taking into account the flight time. Out of the three jumps performed the highest flight time was recorded [22].

Cognitive tests

Focused attention test (Grid)

This instrument allowed the evaluation of outside attention, both broad and narrow according to basic principles of attention control [23]. Its execution consisted of pointing out (according to the instructions provided previously) the numbers 1 to 38 that appeared in a grid with different sized digits, sequentially.

Digit span WAIS subtest (Digits)

Attention, resistance to distraction and immediate auditory memory were measured with a memory function subtest part of Wechsler Adult Intelligence Scale (WAIS) test. It consists of asking subjects to repeat two sets of numbers, which were previously read in direct and reverse order, respectively. A level of nine numbers in direct order and eight numbers in reverse order was used for the test. For this study we used the digit span test by changing the numbers in each sample to avoid learning, but keeping the series, times and original guidelines [24].

These are simple, of short-duration tests, which evaluate cognitive capacities important during the practice of different sports. Both tests have been previously used in similarly designed approaches to the research question of the present work (see [1]).

Statistical analysis