This study aims to analyze in detail selected indicators of appetite regulation, metabolism, and iron balance in competitive rowers undergoing intense exercise. The project involves assessing dynamic hormonal and biochemical changes in the athletes' bodies, which will allow a better understanding of the body's adaptive mechanisms to prolonged physical strain.

Research group: the research will be conducted in a homogeneous group of professional rowers (N=30), all competitors qualified for the Polish Youth Rowing Team:

• aged 19 - 24, because the age group must be homogeneous,
• the size of the group is about thirty people, which in the case of a homogeneous group will enable statistical analyzes to be conducted.

Rowing performance test: At the beginning and end of the training camp, participants will perform a test on a rowing ergometer (Concept II, USA). Each subject will have to cover a distance of 2000 m in the shortest possible time, which is the distance starting in rowing competitions. The results of both tests will be considered in the selection for the championship team; therefore, athletes will be well motivated to perform both tests with maximum effort.

Blood samples will be taken from the cubital vein at three-time points: before each ergometer test (after overnight fasting), directly after the test, and after a 1-hour recovery period.

The study aims to conduct a detailed analysis of selected indicators of appetite regulation, metabolism, inflammation, and iron balance in competitive rowers undergoing intense exercise. The project involves assessing dynamic hormonal and biochemical changes in the athletes' bodies, which will allow for a better understanding of the body's adaptive mechanisms to prolonged physical strain.

Research group: The research will be conducted in a homogeneous group of professional rowers (N=30), all competitors qualified for the Polish Youth. Rowing Team:

• aged 19 - 24, because the age group must be homogeneous,
• the size of the group is about thirty people, which in the case of a homogeneous group will enable statistical analyzes to be conducted.

Rowing performance test: at the beginning and at the end of the training camp, participants will perform a test on a rowing ergometer (Concept II, USA), each subject will have to cover a distance of 2000 m in the shortest possible time, which is the distance starting in rowing competitions. The results of both tests will be considered in the selection for the championship team; therefore, athletes will be well motivated to perform both tests with maximum effort.

Blood samples will be taken from the cubital vein at three-time points: before each ergometer test (after overnight fasting), directly after the test, and after a 1-hour recovery period.

Measurement: All determined parameters will be measured with the available equipment in the biochemical laboratory of Poznan University of Physical Education, Faculty of Sport Sciences in Gorzow Wlkp., using commercial assay kits. Measurements will be performed by the project contractors.

1. Polyethylene tubes (4.9 ml) containing dipotassium ethylenediaminetetraacetic acid (EDTAK2) anticoagulant will be used for the following tests:

   (a) complete blood count (18 parameters) determined on the MYTHIC 18 hematology analyzer (Orphee Medical, Geneva, Switzerland). White blood cell indices: WBC (white blood cells), LYM (lymphocytes as a percentage), LYM (lymphocytes), MON (monocytes as a percentage), MON (monocytes), GRA (granulocytes as a percentage), GRA (granulocytes). Red blood cell indices: RBC (Red Blood Cells), HGB (Hemoglobin), HCT (Hematocrit), MCV (Mean Corpuscular Volume), MCH (Mean Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), RDW (Red Blood cells Distribution Width). Platelet indices: PLT (Platelet), MPV (Mean Platelet Volume), PDW (Platelet Distribution Width), PCT (Thrombocrit) (b) gene expression.

2. Polyethylene clotting activator tubes (9 ml) will be centrifuged to separate the morphotic elements from the serum using a centrifuge (3000 rpm for 10 min). The serum will be pipetted into several Eppendorf tubes, which will then be frozen (temp. -80°C). All the following biochemical parameters will be determined from the extracted serum: using the ELISA method by the test manufacturer's instructions. The designations include the flowing parameters:

   1. Analysis of leptin, ghrelin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY), pancreatic polypeptide (PP) and cholecystokinin (CCK) levels will allow assessment of dynamic hormonal changes in rowers in relation to training intensity, energy availability and diet composition.

      • Leptin (LEP) - a satiety hormone whose levels may be reduced in athletes with low levels of body fat.
      • Ghrelin (GHRL) - a hunger hormone that increases in states of energy deficit and may affect the regulation of caloric intake.
      • GLP-1, PYY, PP and CCK - intestinal peptides involved in appetite suppression and modulation of insulin secretion and fat metabolism.

   2. Biochemical and hormonal parameters

      Stress and recovery hormones:

      • Cortisol - a marker of stress response and catabolism.
      • Testosterone - a key anabolic hormone, whose ratio to cortisol (T/C) helps assess the balance between anabolism and catabolism.

      Neurotransmitters associated with fatigue resistance:

      • Serotonin - important in regulating mood and central fatigue; its excess can contribute to lethargy and decreased motivation.
      • Dopamine - crucial for motivation and mental performance; decreased levels may indicate neurotransmitter depletion.

      Sex hormones and metabolic balance:

      • Estradiol
      • Progesterone - in women indicators of menstrual cycle and hormonal balance,

   3. ferroptosis parameters

      • antioxidants: glutathione (GSH), glutathione peroxidase 4 (GPX4),
      • markers of oxidative stress: 4-Hydroxynonenal (4-HNE), Heme oxygenase 1 (HO-1)
      • iron status parameters: soluble transferrin receptor, ferritin, erythroferrone, lactoferrin

      The iron and Total Iron Binding Capacity (TIBC) levels will be determined using the colorimetric method on the SPECTROstar Nano reader. Unsaturated Iron Binding Capacity (UIBC) will be calculated from the formula: UIBC = TIBC-iron in serum)

   4. Markers of energy metabolism:

      • Glucose - a key energy source for working muscles, to assess how the body regulates carbohydrate metabolism during and after exercise.
      • Glycogen - an important indicator of energy reserve utilization during high intensities, to determine the level of energy reserve utilization.
      • Insulin - responsible for the transport of glucose into muscle cells and to assess how the body manages energy before, during and after exercise, and whether adaptation to load is effective,
      • Lactic acid - an indicator of exercise intensity and the balance between aerobic and anaerobic metabolism.

   Markers of muscle damage:

   • CK (creatine kinase), myoglobin - increases with muscle damage, especially after intense exercise, allows to assess the degree of fatigue and risk of overtraining.
   • interleukin-6 (IL-6) - mediator of inflammatory response and marker of fatigue; rises after intense exercise, indicates fatigue and recovery processes.

   Markers of metabolic adaptation and recovery:

   - fibroblast growth factor-21 (FGF-21) - , a protein, regulates metabolism and restoration of energy resources, allows assessment of the body's adaptation to intense exercise.

3. Using a portable analyzer system for measuring blood gas, electrolytes and metabolites (epoc®), using dedicated epoc® test cards. Measured parameters: Direct: pH, partial pressure of carbon dioxide (pCO2), partial pressure of oxygen (pO2), total plasma carbon dioxide (tCO2), sodium (Na+), potassium (K+), calcium (Ca++), chlorine (Cl-), hematocrit (Hct), glukose (glu), Lactic Acid (Lac), Crea, Urea, urea nitrogen (BUN). Calculated values: cHgb, bicarbonate level (cHCO3), cTCO2, excess base in extracellular fluid (BE ecf), BE(b), total blood saturation (cSO2), glomerular filtration rate (GFRmdr), GFRmdr-a, GFRckd, GFRckd-a, GFRswz, anion gap (AGAP), AGapK, BUN/Crea, Urea/Crea. It will be determined from capillary blood taken from the earlobe (90 µl). Lactic acid will be determined from capillary blood taken from the earlobe (10 µl). The determination will be performed with dedicated reagents, using a portable biochemical photometer Vario Photometer II (Diaglobal, Berlin, Germany).

1. Inclusion Criteria:

   • consent to participate in the study,
   • regular training regimen,
   • current medical examination,
   • no health contraindications (to be decided by a doctor).

2. Exclusion Criteria:

   • Lack of consent for blood draws,
   • injuries,
   • health problems,
   • anti-inflammatory drugs,
   • supportive substances, supplements in the last 3 months before the start of the study.

The Polish Youth Rowing Team