Participants and study design
A simple one-centre study was performed on 12 (1 dropout) healthy males. The inclusion criteria were: age (18–50 years old), sex (male), being a non-smoker, maintaining a balanced diet, and performing physical activity > 3 but < 6 days per week. The sample size was calculated taking into account that a weight loss of 5% was significant with a 95% of confidence. The participants carried out a CR of 30–40% with respect to their usual diet, three alternate days per week for six weeks. Both before and after the every-other-day fasting CR period, the participants took a maximal exercise stress test. For each participant, one blood sample was obtained at the beginning and another at end of the every-other-day fasting intervention at basal conditions and 30 min after acute maximal exercise. All of the participants were informed of the purpose and demands of the study before providing their written consent to participate. The protocol complied with the Declaration of Helsinki for research on human subjects and was approved by the Clinical Research Ethics Committee at the Direcció General de l’Esport of the Catalonian Sports Council. The project was registered at ClinicalTrials.gov (NCT02533479).
All athletes also take part in a previous nutritional intervention study performed in order to evaluate the effects of the energy-balanced diet or dietary supplementation with functional beverages for one month training on physical performance of the athletes [9].
CR prescription
Every participant was interviewed on their dietary, living, and training habits. Daily energy demands were calculated for each participant by taking into account the individual’s resting metabolic rate, body weight, and physical activity per week [10]. The calculated energy demands (mean ± sd) of participants were 2351 ± 156 Kcal/day distributed between a resting metabolic rate of 1862 ± 119 Kcal/day and physical activity of 3426 ± 1147 Kcal/week. Dietary habits were assessed using a 7-day dietary record which tracked all foods and fluids consumed, portion sizes, how foods were prepared, and how consumption habits were distributed throughout the day. Athletes VO2max at the exhaustion was 42.6 ± 1.6 before CR intervention and 40.8 ± 1.1 after CR intervention without any significant difference From this information, a diet analysis was performed using a computer program based on CESNID food composition Tables [11].
Over a period of six weeks, the participants practiced an every-other-day fasting CR program, decreasing calorie intake by 33% with respect to their usual diets. The participants restricted their habitual diets for three alternate days each week and on the other four days participants’ dietary intakes were the same as they were at the beginning of the study without changes to caloric intake or distribution of meals.
Adherence to the nutritional intervention program was assessed using a 7-day dietary record during the last week of the intervention. All foods and fluids consumed, portion sizes, how foods were prepared, and how consumption habits were distributed throughout the day were recorded. From this information a dietary analysis was performed using a computer program based on CESNID food composition Tables [11].
Densitometry
Densitometry was performed by Lunar IDXA (General Electric, USA) following the manufacturer’s details. Cutoffs and references were established according previously published models [12,13,14].
Total body water content
Total body water content was measures by electric impedance using a two frequencies method previously described [15,16,17] .
Exercise stress test energy efficiency
Each subject performed an incremental maximal test until exhaustion on a motorized treadmill (EG2, Vitoria, Spain) in order to determine his maximal oxygen consumption (VO2max) using a computerized metabolic chart (Master Screen CPX,Erich Jaeger, Wuerzburg, Germany). The protocol for the exercise test followed is previously described [9]. Each participant performed a maximal exercise test on a treadmill after fasting overnight at the beginning and again at the end of the nutritional intervention. The athletes spent three consecutive 5-min intervals running at 50%Vmax, 60%Vmax, and 70%Vmax, and then they ran at their upper anaerobic threshold running rate until reaching exhaustion. A continuous calculation of oxygen consumed (mL/min) and CO2 exhaled (mL/min) was made, and energy expenditure per minute (Kcal/min) at each running speed was calculated using Weir’s eq. [18]. Energy expenditure per meter run (Cal/m) was calculated using oxygen consumed (mL/min) and running rates (m/min) in Mora’s eq. [19].
Experimental procedure
Venous blood samples were obtained in basal and 30 min after each exercise test from the antecubital vein of participants with vacutainers containing EDTA (ethylenediaminetetraacetic acid) as anticoagulant (6 mL) to obtain plasma and also to purify erythrocytes following an adaptation of the method described elsewhere [20]. Others venous blood samples were obtained to determine blood cells counts and plasma markers of nutritional status. Erythrocytes counts, hemoglobin, hematocrit, platelets counts, glucose, urea, uric acid, creatinine, bilirubin, calcium, cholesterol total, HDL, LDL, triglycerides, iron, transferrin, transferrin iron, transferrin saturation index, ferritin, vitamin D, the enzymes activities of glutamate, pyruvate transaminase, oxaloacetate transaminase, gamma-glutamyl-transferase, creatine kinase were determined by standardized clinical analytical methods.
Lactate determination
Blood lactate was measured using a microsample of blood (20 μL) was taken from the ear while athletes were performing stress test. Blood samples were obtained at 50%, 60%, 70% of Vmax, and immediately at the end of the last bout to exhaustion (Dr. Lange®, Berlin, Germany).
Malondialdehyde (MDA) determination
MDA levels as marker of lipopeoxidative damage were analyzed using a method previously described [21].
Determination of erythrocyte FAs profile
Erythrocyte fatty acids composition was determined by gas chromatography/mass spectrometry using the method by Lepage and Roy [22] after total lipid extraction using the method of Folk [23] and its transformation to methyl esters by reaction with acetyl chloride. Non-esterified heptadecanoic acid (Nu-Chek Prep, Mn) in hexane was then added as an internal standard and it also containing 0.01% butylhydroxytoluene as an antioxidant. A methyl ester peaks were identified through mass spectra and by comparing the elution pattern and relative retention times of FA methyl esters. The results are expressed in relative amounts (percentage molar of total FAs).
Statistical analysis
Statistical analysis was carried out using the Statistical Package for Social Sciences (SPSS v.21.0 for Windows). Results are expressed as mean ± standard error of the mean (SEM). The statistical significance of the data was assessed by student’s t-test for unpaired data and p < 0.05 was considered statistically significant.