Muscle biopsy remains the gold standard for assessing molecular and structural properties of skeletal muscle, yet conventional techniques such as the current gold standard Bergström method (Bergstrom, 1962) require large tissue samples (≈100–250 mg) and involve skin incisions, limiting their use in vulnerable populations, including clinical cohorts, paediatric patients, and elite athletes who are reluctant to sacrifice substantial amounts of muscle tissue. Also, repeated sampling is highly suboptimal as the procedure itself will induce damage and inflammation (Malm et al., 2000). Fine-needle muscle microbiopsy has emerged as a minimally invasive alternative, enabling sampling with needles of 0.9–2.3 mm diameter and yielding 2–50 mg of tissue, which is sufficient for modern multi-omics workflows. State-of-the-art liquid chromatography mass spectrometry can identify thousands of proteins from a few micrograms of proteins (Murgia et al., 2021), reducing the need for large biopsies.

Despite its promise, most published microbiopsy protocols employ relatively large 13 to 16-gauge (G) needles (Hayot et al., 2005; Townsend et al., 2016; Pietrangelo et al., 2011), which may compromise tolerability and ethical acceptability in sensitive cohorts. Regulatory bodies, including the Swedish Ethical Review Authority, have requested empirical evidence comparing pain perception between muscle microbiopsy and routine venipuncture before approving studies in paediatric or clinical populations. Such data are currently lacking.

Here, we present a refined muscle microbiopsy protocol using a 20-G needle, smaller than those previously reported, and provide evidence of its clinical tolerability relative to standard venous blood sampling. This methodological advance reduces invasiveness while maintaining sample adequacy for downstream proteomic analysis, addressing a critical barrier to broader adoption in research and clinical settings.

A total of 78 microbiopsies were successfully obtained. Pain ratings for the first microbiopsy and venipuncture are summarized in Table 1. Each microbiopsy yielded approximately 2 mg of skeletal muscle tissue.

There was no significant difference in pain between procedures (Z(25) = 1.01, P = 0.311). All participants (100%) reported low pain (VAS ≤3) for microbiopsy, compared to 83% for venepuncture. No adverse events occurred during or following the microbiopsy procedure. While participants were not systematically asked to record pain or discomfort at 24–72 h post-procedure, they were instructed to contact the study team in case of delayed pain, bleeding, hematoma, or other adverse events. All participants initiated supervised whole-body resistance training within a few days after the micro biopsy, and no participant reported post-biopsy pain or discomfort.

The present feasibility study demonstrates that fine-needle muscle microbiopsy of the vastus lateralis using a 20G needle in healthy adults is both feasible and clinically tolerable.

With only a self-applied topical anesthetic cream, most participants reported minimal or no pain, and ratings were comparable to or lower than routine venipuncture. However, we should note that local anaesthetic cream was applied before the microbiopsy, whereas venous blood sampling was performed without analgesia, as is standard practice. This difference may influence direct pain comparisons. Also, the venipuncture used a 21-G needle, which has a smaller outer diameter than the 20-G needle used for the microbiopsy, and therefore may inherently cause less discomfort. Future studies should consider standardized analgesia or include a no-anaesthetic microbiopsy condition to isolate intrinsic pain levels.

Although not directly examined in the present study, reported pain levels are markedly lower than those typically reported for standard Bergström biopsies, which require injection of local anesthetic and are commonly rated in the 4–5 range on a 0–10 scale (Hayot et al., 2005; Dengler et al., 2014). Beyond improved tolerability, the fine-needle approach substantially reduces tissue burden while providing sufficient material (1–2 mg) w.w. muscle tissue, which is well enough for advanced multi-omics analyses using tandem mass spectrometry. Yet, it should be noted that using this small of a biopsy needle do not provide sufficient material for traditional immunohistochemistry methods, including fibre-type composition analyses. However, this is possible using mass-spectrometry, which requires only microgram quantities of protein (Murgia et al., 2021).

A limitation in this study is that delayed soreness or discomfort at (i.e. 24–72 h) was not systematically recorded. While no delayed adverse events were reported to the research team, the absence of structured follow-up limits conclusions regarding short-term post-procedural soreness or localized inflammatory responses. Future studies should include standardized follow-up measures. Procedural success, complication rates, and perceived pain may be influenced by operator experience with both microbiopsy and venipuncture. In the present study, procedures were performed by trained personnel; however, we did not quantify the effect of operator expertise on pain outcomes. Further, one should consider further standardization (e.g., insertion depth, and where feasible ultrasound guidance) to minimize variability and enhance safety, which may be particularly pertinent in paediatric cohorts.

We did not record tissue mass at the time of sampling because the protocol prioritized rapid cryopreservation to preserve labile metabolites. As a result, we cannot report intra-subject variance in tissue yield across replicates in this manuscript. Nevertheless, on the basis of core dimensions and prior protocol experience, each biopsy provided >1 mg of tissue as documented in pilot biopsies, which is well above the minimum input needed for modern proteomics such as UHPLC-LC/MS-MS.

These findings address a key ethical and practical concern for applying muscle biopsy in vulnerable populations and support integration of this technique into longitudinal and interventional research.

Future work should formally quantify biopsy mass and protein content, establish reproducibility regarding protein- and gene-expression, sample integrity for multi-omics applications, and feasibility in paediatric and clinical cohorts. By combining minimal invasiveness with good tolerability, the 20G fine needle microbiopsy shows potential for broader research and clinical application.