Abstract：Human skeletal muscle plays a vital role in maintaining quality of life by enabling mobility that facilitates experiencing the world. Early animal studies demonstrated the extraordinary reparative and regenerative capabilities of skeletal muscle in response to various damaging challenges. These findings prompted the introduction of muscle biopsy techniques in human exercise science research. This approach has significantly advanced our understanding of how human muscles adapt to physical challenges and derive health benefits from exercise. To date, over 10,000 peer-reviewed articles have been published using this invasive technique. Muscle biopsies have been instrumental in uncovering insights into how muscles alter gene expression to adapt to stress, how bone marrow-derived cells contribute to muscle tissue renewal, and how energy substrates are utilized during and after exercise. This has significantly advanced our knowledge in exercise science. In 1965, Jonas Bergström designed the first muscle biopsy needle and applied it to exercise science research. His initial studies were published in Acta Medica Scandinavica and Acta Physiologica Scandinavica in 1966 and 1967 (Ahlborg et al., 1967; Bergström & Hultman, 1966). He recruited nine healthy participants to undergo muscle biopsies before completing a glycogen-depleting exercise at 75% VO_(2max). Following this, participants consumed one of three diets with equal caloric content: a mixed diet, a fat ± protein diet, or a carbohydrate-rich diet, in a crossover design. Bergström discovered that endurance time was significantly higher (189 minutes) following the high-carbohydrate diet, due to greater muscle glycogen content, compared to the fat-protein diet (59 minutes) and the mixed diet (126 minutes). This breakthrough established the critical role of carbohydrates as the primary macronutrient for human endurance performance. Evans et al. (1982) subsequently proposed an improved muscle biopsy technique, published in the Medicine & Science in Sports & Exercise. This method recommends the use of vacuum-assisted biopsy to obtain larger muscle samples. Medical diagnostic results from human muscle tissue samples enhance doctors' precision in diagnosing diseases of the nervous system, connective tissue, vascular system, and musculoskeletal system. In clinical medicine, muscle biopsy involving needle biopsy and punch biopsy to obtain muscle tissue is a vital technique for diagnosing various muscle diseases such as muscular dystrophy, polymyositis, and myofibrillar myopathy in humans. Needle biopsy involves inserting a thin needle into the muscle to extract a small tissue sample. Open biopsy requires making a small incision to directly remove a larger piece of muscle tissue (Ray-Coquard et al., 2003). Core needle biopsy utilizes a special hollow needle to obtain a larger cylindrical tissue sample through the skin and muscle layers (Bruening et al., 2010). Vacuum-assisted biopsy combines vacuum technique with a larger needle to repeatedly extract multiple tissue samples through a single puncture (Skrzynski et al., 1996). There will be varying usage requirements depending on medical examination needs, along with their respective advantages and disadvantages, as shown in Table 1 (Kasraeian et al., 2010). Local anesthetics like lidocaine are usually administered to minimize discomfort during the procedure. In our laboratory, 2% lidocaine is typically used at the biopsy site, with doses ranging from 1 ~ 2 ml depending on the participant's needs to ensure effective anesthesia. Muscle biopsy samples allow detailed analysis of muscle structure and function through methods like periodic acid-Schiff (PAS) stain for glycogen concentration, reverse transcription polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. In recent years, our team has collaborated with a physician to explore ergogenic aids in athletic competitions, including nutritional approaches with varied glycemic indexes for improved glucose uptake, effects on muscle glycogen replenishment, and impacts on mitochondrial biogenesis (Cheng et al., 2024; Huang et al., 2020). In aging research, muscle biopsy technique has revealed a high number of aging stem cells in the muscles of young adults and demonstrated that moderate-intensity aerobic exercise alone may not adequately clear senescent cells (Wu et al., 2019). These findings represent significant advances in our understanding of muscle physiology. Muscle biopsy technique, initiated by Bergström in 1965 and later improved by Evans et al. in 1982, has also been widely used in exercise and sports science. Applications include studies on muscle physiology, sports performance, exercise-induced muscle damage, muscle energy substrates during exercise, and mitochondrial biogenesis (Figure 1). For example, Iaia et al. (2011) demonstrated that high-intensity exercise performance is associated with fast-twitch muscle fibers, while muscle capillaries and the Na+-K+ pump β1-subunit influence performance in shorter, intense activities. In addition, muscle biopsy has been utilized to study alternative exercise training methods, such as blood flow restriction (BFR) training. Christiansen et al. (2019) conducted a six-week BFR training study that revealed significant changes in muscle glucose utilization and antioxidant enzyme activity in the thigh muscles. Other researchers have found that BFR training increases muscle vascular permeability and free radical production, leading to physiological adaptations (Bennett & Slattery, 2019; Christiansen et al., 2019). Most studies in exercise and sports sciences that use biopsy protocols (about 92%) focus on the vastus lateralis muscle group (Newmire & Willoughby, 2022). The vastus lateralis muscle is commonly chosen because it has a significant muscle mass, is easily accessible, and has a low density of neurovascular branches, which minimizes the risk of complications. Moreover, the vastus lateralis is actively engaged in typical sports stances, making it an ideal muscle for studies aiming to represent whole-body movement. These anatomical and functional considerations support its use for muscle biopsy (Chen et al., 2019). Emphasizing the importance of preventing adverse events during muscle biopsy procedures, Chen et al. (2019) identified the safest biopsy site as the area just anterior to the lateral fascia (iliotibial band), between 25% ~ 50% of the distance from the lateral joint line to the greater trochanter. Some studies also target other muscles, such as the gastrocnemius, soleus, latissimus dorsi, triceps brachii, lumbar multifidus, erector spinae, obliques, and upper trapezius. (The form abridges) In Taiwan, the adoption of muscle biopsy technique has expanded, offering a valuable research tool for exercise science. Although muscle biopsy methods have evolved to reduce invasiveness, psychological and physical stress for participants remains a concern. To address this, we have adopted a modified biopsy approach using two 14-gauge needles to increase sample size while minimizing trauma (Figure 2). This technique improves sample collection efficiency and participant comfort, making it more feasible for human studies compared to traditional methods like open biopsy, core needle biopsy, and vacuum-assisted biopsy. By continuing to develop and refine muscle biopsy methods, we aim to support sports science research in Taiwan and facilitate studies on exercise training, supplements, and health strategies. (Full text)