Purpose The purpose of this study was to determine the peak point of irisin following exercises of different intensities.

Method This randomized crossover controlled trial aimed to examine the acute effects of three experimental conditions-high-intensity interval training, long-interval continuous training, and low-intensity continuous training-on serum irisin and blood lactate levels in healthy young adults aged 18-25 years.

All participants completed the three conditions in a randomized and counterbalanced manner. To ensure balanced exposure, they were assigned to one of three groups (n = 4 per group), each following a distinct condition order based on a Latin square design. This approach guaranteed that each session occurred equally across time points (sessions 1-3) and that the order minimized sequence effects by rotating the position of each condition relative to the others.

To control for potential carryover-particularly from elevated endocrine responses following intense exercise (increases in irisin had been reported)-no two high-intensity sessions were scheduled consecutively. Where possible, higher- and lower-intensity protocols were alternated. Furthermore, a seven-day interval separated each session to reduce residual physiological or cognitive effects.

The trial protocol was developed in line with CONSORT and SPIRIT guidelines, including relevant extensions for within-subject designs and non-pharmacological interventions. Participants were blinded to the study hypothesis.

All exercise sessions were conducted in the morning (09:00-11:00) on a track and field pitch under the supervision of a certified strength and conditioning coach to ensure consistency and adherence to protocol. Participants were instructed to avoid strenuous activity for 48 hours prior, abstain from alcohol and caffeine for 24 hours, and obtain at least 7-8 hours of sleep before each session. Dietary habits and general lifestyle were kept unchanged throughout the study.

During the first visit, participants' body weight and body fat percentage were assessed using bioelectrical impedance analysis. Resting heart rate was then recorded, followed by administration of the Yo-Yo Intermittent Recovery Test Level 1 to determine maximal aerobic speed (MAS), aerobic capacity, and peak heart rate.

In the following four visits, each participant completed all experimental conditions. Each exercise session began with a standardized 10-minute warm-up, including light jogging, dynamic stretching, and movement preparation drills.

All exercise sessions were conducted on a standard track and field facility and lasted 40 minutes in total, consisting of a 10-minute standardized warm-up, 24 minutes of exercise, and a 5-minute recovery period. The exercise component was individually prescribed based on each participant's MAS, determined from the Yo-Yo Intermittent Recovery Test Level 1. All running distances were calculated individually using time × speed formulas based on each participant's MAS, ensuring workload equivalence across conditions.

High-Intensity Interval Exercise (HIIE): Participants ran at 105-110% of their MAS for 15-second intervals, covering a pre-calculated distance during each bout. After each 15-second sprint, they rested passively for 15 seconds and returned to their starting point. This was repeated continuously for 5 minutes, constituting one set. A total of four 5-minute sets were completed, interspersed with 2.5-minute passive rest periods, yielding a total exercise time of 30 minutes.

Moderate-Intensity Continuous Exercise (MICE): Participants ran continuously for 30 minutes at 70% of their MAS without rest.

Low-Intensity Continuous Exercise (LICE): Participants ran continuously for 30 minutes at 50% of their MAS without rest.

Inclusion Criteria:

• Aged between 18 and 25 years
• No known medical condition that prevents participation in exercise
• Not currently using any pharmacological agents

Exclusion Criteria:

• Experiencing any discomfort before, during, or after exercise
• Current or past diagnosis of pharmacological or doping substance use
• Engaging in regular exercise (more than 3 times per week) within the last 2 weeks
• History of alcohol or substance addiction
• Any other condition or factor that may prevent full participation in the study protocol

Only male participants are eligible to participate in this study based on biological sex.