**Method**

#### **Participants**

Young non-athletic males (20. 9 ± 0. 7 years) were selected from the total number of participants based on their characteristics, including stature, weight, physical activity level and tissue- Maximum isostatic knee extension torque at 45° knee flexion. The level of physical activity was determined during an interview to determine the amount and type of activity of the participants. All participants volunteered and gave their written consent to participate in this study, a process approved by the Medical Ethics Committee.

#### **Muscle Biopsies:**

Using needle aspiration biopsy, we first collected a sample of Vastus lateralis from the participant's left leg. Prior to the biopsy, all participants were informed to abstain from strenuous physical activity. A small portion of muscle biopsies was inserted into TissueTek and frozen in nitrogen-cooled isopentane for immunohistochemical analysis. It was previously described that for myosin heavy chain isoforms, muscle infection was stained by immunohistochemistry. Samples used for the single-strand test were immediately placed at in cold stripping solution (0°C) and a bundle of filaments was cut lengthwise. At −20° C, packet skin peel solution was stored as a regular replacement for at least 5 days before the first experiment.

#### **Single Muscle Fiber Experiments:**

After counting, individual muscle fibers were tested for maximum trigger force normalized for fiber cross-sectional area (P0), maximum no-load velocity (V0), and force-velocity relationship. or passive tension properties on dynamic force profiles. Within 4 weeks after biopsy, all experiments were performed at 15°C. within four weeks after biopsy. Detailed protocols have been described previously. Briefly, after adjusting the length of the sarcomere to 2.5 m, the size of an optical fiber including the fiber length and the cross-section (CSA) was determined on the digital image of the optical fiber. When damaged fibers were removed prior to testing to exclude an effect on the contractile properties of the fibers, each fiber was visually examined for damage by shearing or processing of muscle biopsy results.. P0 was determined as the maximum stabilizing force developed by the fiber when immersed in the activator solution (pCa 4.5) corrected for the CSA fiber. By slack test, no-load shortening rate (VO) was measured; each fiber is maximally activated and then rapidly shortened, so that force drops to baseline and re-evolves after a period proportional to stride length. in the number of serial sarcomas between different fibers, the V0 fiber was determined as the slope of the fitted line and expressed as fiber length per second (FL/s) to account for the difference tested. The yarn was subjected to 5–6 times three consecutive isometric load clamps to determine the force-velocity relationship. Based on Hill's equation, the data obtained on an optical fiber are adjusted using an iterative nonlinear curve tuning procedure (Marquardt Levenberg algorithm). From the parameters of the adjusted forcevelocity curve relationship, the fiber power (W/L) was determined. Passive tension was tested with a progressive stretch protocol while the fiber was still in pCa 9.0 solution. To evaluate the elastic properties of the fibers, Young's modulus (kN/m2) and hysteresis (kN/m2) were calculated & After mechanical testing was completed, the fiber fragment was dissolved in 25 μl sample buffer SDS and stored at −20°C until MHC isoform content analyzed by SDSPAGE.

## **Genotyping**

In, using the manufacturer's procedure I Chemo magnetic Separation Module (PerkinElmer, Baesweiler, Germany), DNA was extracted from an EDTA blood sample at UZ Leuven. In this experiment, the TaqMan SNP genotyping assay, ACTN3 R577X polymorphism genotyping (rs. 1815739) was performed (ID C\_\_590093\_1, Applied Biosystems). Here, real-time qPCR was performed in 20 L of reaction mixture with 1 L of DNA, and other equipment required for the experiment was 8 L of RNase-free water, 1 L 20 × test mixture of TaqMan SNP genotyping and 10 μl of 2 × Taqman Universal PCR main combination (Applied Biosystems).

#### **Statistical analysis**

Using Student's t-test, participant characteristics and a muscle fiber composition were compared between ACTN3 577RR and 577XX individuals. An ANOVA analysis in SAS version 9.2 (SAS Institute, Cary, NC) measured single strands per fiber between the two groups of genotypes. A mixed process with a multilevel model is used to explain the dependence of multiple fibers in a single participant. For genotype differences, unbiased effect sizes and 95% confidence intervals are reported. Only Class I and Class IIa fibers are sufficiently abundant for a statistical analysis [33].
