This clinical study evaluates the effect of 500 mg of 2S-hesperidin for 8 weeks on performance (power generated in different metabolic zones), body composition (fat and muscle mass) and biochemical (antioxidant, inflammatory status) and metabolic (capillary blood in finger) markers in amateur cyclists. Our hypothesis is that chronic intake of 2S-hesperidin can improve performance (maximum power generated). To justify this hypothesis, we measured the parameters mentioned above, which could establish a cause-effect relationship between 2S-hesperidin intake and possible yield improvement.
Participants All participants were informed about the procedures and provided signed informed consent. The study was conducted according to the guidelines of the Helsinki Declaration for Human Research and the protocol was approved by the Ethics Committee/Institutional Review Catholic University of Murcia (Code: CE091802). Study design Cyclists were instructed to take the supplement along with breakfast and to continue their usual diet and training schedule. Subjects in both groups were instructed not to consume foods high in citrus flavonoids (grapefruit, lemons or oranges) for 5 days prior to and during the study, this was verified by diet recalls records. Procedures Participants visited the laboratory on seven occasions. Visit 1 consisted of a medical examination and blood extraction to determine health status. On visits 2 and 5, a 24-hr diet recall and a Wingate test were performed. On visits 3 and 6, another 24-hour diet recall was conducted, followed by an incremental test until exhaustion on a cycle ergometer. On visits 4 and 7, the 24-hour diet recall was repeated, and participants performed a rectangular test on the cycle ergometer. Prior to each testing session (visits 2, 3, 4, 5, 6 and 7), a standardized breakfast composed of 95.16 g of carbohydrates (68%), 18.86 g of protein (14%) and 11.30 g of lipids (18%) was prescribed by the sport nutritionist. Pre and post evaluation test Pre-intervention visits: 1,2,3 and 4 Post-intervention visits: 5,6 and 7 Visit 1: Health status blood analysis: A general analysis will be performed both in the pre and post-supplementation period. The extraction will be done by venous via. (Fasting) Medical examination: A clinical history of family and personal history, a resting electrocardiogram (ECG) and a medical examination (auscultation, blood pressure measurement, etc.) will be taken to certify that the person is healthy and not at risk to participate in the study. (Fasting) Visits 2 and 5: Resting Metabolic Rate (RMR): A resting test will be performed with the ergospirometer to determine macronutrient supply at rest and caloric expenditure. (Fasting) Body composition by absorptiometric densitometry (DXA): A body composition test will be performed by the dual energy absorptiometric densitometry technique (X-ray), with the objective of to determine parameters of fat mass, fat-free mass and fat percentage. In addition, anthropometry was also used to determine changes in body composition (fat and muscle mass). (Fasting) Anaerobic power and mechanical power production: After a standardized breakfast, a Wingate test was performed on a cycloergometer. Wingate test (WAnT) consisted of an all-out 30-s sprint on a cycloergometer (Monark Ergomedic 894E Peak Bike, Vansbro, Sweden). Breaking resistance was held constant at 7.5% of each individual's body mass. All participants were verbally encouraged to pedal as fast as possible during the entire sprint. Visits 3 and 6: Incremental step with final ramp test was performed on a cycle ergometer using a metabolic cart (Metalyzer 3B. Leipzig, Germany) to determine maximal fat oxidation zone (FatMax), ventilatory thresholds 1 (VT1) and 2 (VT2) and maximal oxygen consumption (VO2max). Participants began cycling at 35W for 2 min, increasing then by 35W every 2 min until RER\>1.05, initialling then the final ramp (+35W·min-1) until exhaustion. The estimated functional threshold power (FTP) was calculated using the following equation \[16\]: FTP (W) = Pmax (W) x 0.865 - 56.484. Visits 4 and 7: Rectangular test was performed on a cycle ergometer using the power output values resulting from the maximal test (FatMax, VT1 and VT2). Participants exercised continuously from FatMax, to VT1 and to VT2 for 10 min, without rest. Cardiorespiratory variables (VO2, VO2R, carbohydrate oxidation (CHO), fat oxidation (FAT) and cycling economy) were determined for each metabolic zones. Urine samples Main hesperidin metabolites were analysed in participants' urine. Urine samples, corresponding to 24 h urine collection from each participant before and after the supplementation, were frozen in liquid nitrogen after collection and thawed for its analysis. All the data of the subjects who participated in the study were stored under a computer system with a security key. In addition, a record was also made of some of the paper data that was saved under key, and to which only the researchers of this project had access.
Study Type
INTERVENTIONAL
Allocation
RANDOMIZED
Purpose
TREATMENT
Masking
TRIPLE
Enrollment
40
Cardiose® is a natural orange extract that due to its unique manufacturing process, maintains most of the natural hesperidin isomeric form (2S)
Microcellulose was used, this molecule was selected to avoid affecting the glycemia, as it can occur with dextrose or maltodextrin.
Research Center for High Performance Sport. Catholic University of Murcia
La Ñora, Murcia, Spain
Maximum power
During the incremental test until exhaustion, the power generated was measured maximum power (W)
Time frame: Throughout study completion, an average of 8 weeks
Power generated at FatMax
During the incremental test until exhaustion, the power generated was measured in FatMax (W)
Time frame: Throughout study completion, an average of 8 weeks
Power generated at ventilatory threshold 1 (VT1)
During the incremental test until exhaustion, the power generated was measured in VT1 (W)
Time frame: Throughout study completion, an average of 8 weeks
Power generated at ventilatory threshold 2 (VT2)
During the incremental test until exhaustion, the power generated was measured in VT2 (W)
Time frame: Throughout study completion, an average of 8 weeks
Fat mass
Fat mass (g) was measured by densitometry and anthropometry.
Time frame: Throughout study completion, an average of 8 weeks
Sum of 8 skinfolds
By means of anthropometry, the sum of 8 skinfolds (mm) was calculated.
Time frame: Throughout study completion, an average of 8 weeks
Muscle mass
Muscle mass (g) was measured by densitometry and anthropometry
Time frame: Throughout study completion, an average of 8 weeks
Resting metabolic rate
Resting metabolic rate was measured by indirect calorimetry.
Time frame: Throughout study completion, an average of 8 weeks
Superoxide dismutase (antioxidant marker)
During the rectangular test, superoxide dismutase (U/g Hb) was measured in venous blood.
Time frame: Superoxide dismutase was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Catalase (antioxidant marker)
During the rectangular test, catalase (U/g Hb) was measured in venous blood.
Time frame: Catalase was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Reduced glutathione (antioxidant marker)
During the rectangular test, reduced glutathione (GSH) (nmol/mg protein) was measured in venous blood.
Time frame: Reduced glutathione was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Oxidized glutathione (antioxidant marker)
During the rectangular test, oxidized glutathione (GSSG) (nmol/mg protein) was measured in venous blood.
Time frame: Oxidized glutathione was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
GSSG/GSH ratio (antioxidant marker)
During the rectangular test, GSSG/GSH ratio (%) was measured in venous blood.
Time frame: GSSG/GSH ratio was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Hemoxigenase 1 (antioxidant marker)
During the rectangular test, hemoxigenase 1 (ng/mL) was measured in venous blood.
Time frame: Hemoxigenase 1 was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Thiobarbituric acid reactive substances (oxidant marker)
During the rectangular test, thiobarbituric acid reactive substances (TBARS) (mcm/L) ( are formed as a byproduct of lipid peroxidation) was measured to determine the oxidative state in venous blood.
Time frame: Thiobarbituric acid reactive substances was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Interleukin 6 (inflammatory marker)
During the rectangular test, interleukin 6 (pg/ml) was measured in venous blood.
Time frame: Interleukin 6 was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Tumor necrosis factor (inflammatory marker)
During the rectangular test, tumor necrosis (pg/ml) was measured in venous blood.
Time frame: Tumor necrosis factor was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
C-reactive protein (inflammatory marker)
During the rectangular test, C-reactive protein (mg/L) was measured in venous blood.
Time frame: C-reactive protein was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Monocyte chemoattractant protein-1 (inflammatory marker)
During the rectangular test, monocyte chemoattractant protein-1 (pg/ml) was measured in venous blood.
Time frame: Monocyte chemoattractant protein-1 was measured in pre, after the maximum power phase of the rectangular test and after the recovery phase in pre and post intervention (8 weeks) in both groups.
Hemoglobin (oximetry marker)
During the rectangular test, hemoglobin (g/dL) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Hemoglobin was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Hematocrit (oximetry marker)
During the rectangular test, hematocrit (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Hematocrit was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Oxygen saturation (oximetry marker)
During the rectangular test, oxygen saturation (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Oxygen saturation was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Oxygen partial pressure (oximetry marker)
During the rectangular test, oxygen partial pressure (mmHg) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Oxygen partial pressure was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Carbon dioxide partial pressure (oximetry marker)
During the rectangular test, carbon dioxide partial pressure (mmHg) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Carbon dioxide partial pressure was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Oxyhemoglobin (oximetry marker)
During the rectangular test, oxyhemoglobin (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Oxyhemoglobin was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Carboxyhemoglobin (oximetry marker)
During the rectangular test, carboxyhemoglobin (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Carboxyhemoglobin was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Deoxyhemoglobin (oximetry marker)
During the rectangular test, deoxyhemoglobin (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Deoxyhemoglobin was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Methemoglobin (oximetry marker)
During the rectangular test, methemoglobin (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Methemoglobin was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Total blood oxygen concentration (oximetry marker)
During the rectangular test, total blood oxygen concentration (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Total blood oxygen concentration was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Total blood carbon dioxide concentration (oximetry marker)
During the rectangular test, total blood carbon dioxide concentration (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Total blood carbon dioxide concentration was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Oxygen partial pressure at 50% oxygen saturation (oximetry marker)
During the rectangular test, oxygen partial pressure at 50% oxygen saturation (mmHg) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Oxygen partial pressure at 50% oxygen saturation was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Relative physiological Shunt (oximetry marker)
During the rectangular test, relative physiological Shunt (%) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Relative physiological Shunt was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Alveolar-arterial gradient (oximetry marker)
During the rectangular test, alveolar-arterial gradient (mmHg) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Alveolar-arterial gradient was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Bicarbonate (metabolic marker)
During the rectangular test, bicarbonate (mmol/L) wasa measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Bicarbonate was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Glucose (metabolic marker)
During the rectangular test, glucose (mg/dL) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Glucose was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Lactate (metabolic marker)
During the rectangular test, lactate (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Lactate was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
pH (metabolic marker)
During the rectangular test, pH was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: pH was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Actual base excess (metabolic marker)
During the rectangular test, actual base excess (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Actual base excess was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Standard base excess (metabolic marker)
During the rectangular test, standard base excess (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Standard base excess was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Sodium (metabolic marker)
During the rectangular test, sodium (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Sodium was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Potassium (metabolic marker)
During the rectangular test, potassium (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Potassium was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Chloride (metabolic marker)
During the rectangular test, chloride (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Chloride was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
Calcium (metabolic marker)
During the rectangular test, calcium (mmol/L) was measured using capillary blood obtained from a finger prick and analyzed in a blood gas analyzer (ABL90 FLEX PLUS, Radiometer Iberica, Spain)
Time frame: Calcium was analyzed pre, at the end of the FatMax phase, VT1, VT2, maximum power, FatMax2 and after the recovery phase of the rectangular test in pre and post intervention (8 weeks) in both groups.)
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