Aim of the Study The aim of this study is to evaluate the acute effects of walking and cycling on serum physiological biomarkers associated with autophagy (Beclin-1, LC3, and ATG3).
Previous studies have mostly investigated changes in biomarker levels following a single type of exercise. Therefore, the effects of exercise regimens with different mechanical loading characteristics on circulating autophagy-related biomarkers are not sufficiently understood.
This study aims to comparatively examine the acute effects of two different exercise regimens-walking and cycling-on serum biomarkers associated with autophagy.
Hypotheses H0: There is no change in serum Beclin-1, LC3, and ATG3 levels following walking and cycling.
H1: There is a change in serum Beclin-1, LC3, and ATG3 levels following walking and cycling.
Inclusion Criteria Healthy male volunteers aged 18-35 years Individuals who engage in physical activity at least 30 minutes per day, 2-3 days per week Individuals without any known health problems
Exclusion Criteria Individuals with acute or chronic injuries in the unilateral/bilateral lower extremities Individuals with musculoskeletal disorders in the unilateral/bilateral lower extremities Individuals who have undergone surgical intervention involving the hip and/or knee joints Individuals with cardiopulmonary conditions that would prevent them from performing exercise
Research Method The study will commence after obtaining ethical approval. A total of 20 healthy male volunteers aged between 18-35 years, who are physically active (at least 30 minutes, 2-3 times per week) and have no known health problems, will be included.
To avoid the effects of prior medical conditions, individuals with acute or chronic lower extremity injuries, musculoskeletal disorders, or a history of hip and/or knee surgery will be excluded.
Participants will be instructed to minimize all sports activities 48 hours prior to the assessment sessions; however, normal daily activities such as walking and stair climbing will be permitted. They will also be advised to maintain their usual daily routines and consume the same types of food on evaluation days.
Body weight, body mass index (BMI), age, and heart rate will be recorded before and after the exercise sessions. Exercise intensity will be determined based on heart rate (HR), calculated as:
Lower limit: HR = (220 - age) × 50% Upper limit: HR = (220 - age) × 85% \[8\]
Heart rate will be measured by placing the index and middle fingers on the carotid artery for 15 seconds, and the value will be multiplied by four. The 30-minute exercise protocol will consist of:
First 5 minutes: lower HR limit Next 20 minutes: upper HR limit Final 5 minutes: lower HR limit Anthropometric Measurements Body weight will be measured using a digital scale with 0.1 kg sensitivity, and height will be measured using a digital stadiometer. Participants will be barefoot, wearing only shorts, with knees fully extended, heels together, and standing upright.
Exercise Protocol
Data collection will be conducted in two separate 30-minute exercise sessions, spaced one week apart:
Session 1: Participants will walk on a treadmill at the target heart rate for 30 minutes.
Session 2: One week later, participants will perform 30 minutes of cycling on a bicycle ergometer at a similar heart rate.
All procedures, except for the type of exercise, will be identical between sessions. Participants will attend the laboratory at the same time of day to avoid diurnal variations. A minimum one-week rest period will be provided between sessions to minimize carryover effects.
Blood Sampling Procedure A permanent venous catheter will be inserted into the cubital fossa of either arm. To minimize the effects of prior physical activity, participants will rest in a supine position for 30 minutes upon arrival.
Blood samples will be collected at the following time points:
After the 30-minute rest period (baseline) Immediately after the 30-minute exercise session 30 minutes post-exercise 60 minutes post-exercise The same procedure will be repeated in the second session (cycling). Collection and Analysis of Blood Samples Peripheral blood samples will be collected into gel-containing biochemistry tubes. Approximately 2-2.5 mL of blood (about half a standard tube) will be collected each time.
After clotting at room temperature (20-30 minutes), samples will be centrifuged at 3500 rpm for 10 minutes at +4°C to obtain serum. Serum samples will be stored at -80°C until analysis.
Protein Measurement Serum protein levels will be measured using ELISA kits according to the manufacturer's instructions. A Multiskan Go spectrophotometer (Thermo Fisher Scientific, Finland) will be used. Measurements will be analyzed using Thermo Scientific SkanIt software, and protein concentrations will be determined by comparison with standard curves.
Analyzed Proteins Beclin-1 LC3 ATG3
