Physical exercise (walking and cycling) is a potent physiological stimulus that simultaneously alters energy balance, mechanical loading, and metabolic demands in the organism. Autophagy is recognized as a fundamental mechanism in the regulation of acute cellular responses to such stimuli, playing a critical role in maintaining cellular homeostasis, removing damaged proteins and organelles, and ensuring metabolic adaptation [1]. Experimental and translational studies have demonstrated that particularly moderate-intensity and controlled mechanical loading can activate autophagic pathways, thereby supporting structural and functional adaptation in muscle, bone, and connective tissues [2-4].

In the current literature, the relationship between autophagy and exercise has largely been evaluated through experimental animal models and a limited number of human studies [5]. Although animal studies have clearly shown that physiological loading such as walking and running increases molecular signals associated with autophagy, the direct assessment of autophagy at the tissue level in humans is limited due to ethical and feasibility concerns, as it requires invasive methods (e.g., muscle biopsy) [3]. Therefore, recent human studies have increasingly focused on indirect evaluation of autophagy through peripheral blood mononuclear cells and circulating biomarkers [6].

Indeed, recent human studies have reported that proteins associated with autophagy may exhibit changes in peripheral blood cells or circulation in response to acute exercise, and that this response may vary depending on the type, intensity, and mechanical characteristics of the exercise [6]. These findings suggest that autophagy is not merely a tissue-specific process but can also be monitored at the systemic level as part of physiological adaptation [7]. However, there is a limited number of human studies that comparatively investigate the acute effects of different exercise modalities with distinct mechanical loading profiles (such as walking and cycling) on serum biomarkers related to autophagy.

In this context, the present project aims to evaluate the acute effects of two common aerobic exercise modalities with different mechanical loading characteristics-walking and cycling-on serum biomarkers associated with autophagy in healthy male individuals. In this study, Beclin-1, LC3, and ATG3 levels will be considered not as direct indicators of autophagic flux in tissues, but as circulating biomarkers associated with the initiation and maintenance of autophagy. By examining changes in the serum levels of these proteins following acute exercise, it is aimed to obtain indirect yet biologically meaningful data regarding exercise-induced cellular adaptation mechanisms without the need for invasive procedures.

In conclusion, this study aims to contribute to an important gap in the current literature by safely and ethically demonstrating the acute physiological responses related to autophagy in humans across exercise modalities with different mechanical characteristics, thereby enhancing our understanding of the exercise-autophagy relationship.

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

Inclusion Criteria:

• Healthy male volunteers aged 18-35
• Engaging in physical activity at least 2-3 days per week for a minimum of 30 minutes
• Having no known health problems

Exclusion Criteria:

• Individuals with acute or chronic injuries in the bilateral/unilateral lower extremities
• Individuals with musculoskeletal disorders in the bilateral/unilateral lower extremities
• Individuals who have undergone surgical intervention to the hip and/or knee joint
• Individuals with cardiopulmonary problems that would prevent them from performing exercise

HEALTY MALE

A total of 20 healthy male volunteers aged between 18 and 35 years, who engage in physical activity at least 2-3 days per week for a minimum of 30 minutes and have no known health problems, will be included in the study.

To avoid the effects of previous medical conditions, individuals with acute or chronic injuries in the bilateral/unilateral lower extremities, musculoskeletal disorders, or a history of surgical intervention to the hip and/or knee joints will be excluded from the study.

Participants will be instructed to minimize all sports activities 48 hours prior to the assessment days; however, normal daily activities such as walking and stair climbing will be permitted. In addition, participants will be advised to maintain their usual daily routines and to consume the same types of food on the assessment days.