Human skeletal muscle characteristics such as fiber type composition, fiber size, and myonuclear content are widely studied in clinical and sports-related contexts. Being aware of the methodological and biological variability of the characteristics is a critical aspect in study design and outcome interpretation, but comprehensive data on the variability of morphological features in human skeletal muscle are currently limited. Accordingly, in the present study, m. vastus lateralis biopsies (10 per subject) from young and healthy individuals, collected in a systematic manner, were analyzed for various characteristics using immunohistochemistry (n = 7) and SDS-PAGE (n = 25). None of the analyzed parameters, fiber type % (FT%), type I and II fiber cross-sectional area (fCSA), percentage fiber type area (fCSA%), myosin heavy chain composition (MyHC%), type IIX content, myonuclear content, or myonuclear domain, varied in a systematic manner longitudinally along the muscle or between the two legs. The average within-subject coefficient of variation for FT%, fCSA, fCSA%, and MyHC% ranged between 13% and 18% but was only 5% for fiber-specific myonuclear content, which reduced the variability for myonuclear domain size to 11%–12%. Pure type IIX fibers and type IIX MyHC were randomly distributed and present in <24% of the analyzed samples, with the average content being 0.1% and 1.1%, respectively. In conclusion, leg or longitudinal orientation does not seem to be an important aspect to consider when investigating human vastus lateralis characteristics. However, single muscle biopsies should preferably not be used when studying fiber type- and fiber size-related aspects, given the notable sample-to-sample variability. NEW & NOTEWORTHY This study provides a comprehensive analysis of the variability of key human skeletal muscle fiber characteristics in multiple sites along and between the m. vastus lateralis of healthy and active individuals. We found a notable but nonsystematic variability in fiber type and size, whereas myonuclear content was distinctively less variable, and the prevalence of type IIX fibers was random and very low. These data are important to consider when designing and interpreting studies including m. vastus lateralis biopsies.

We have recently demonstrated performance-enhancing effects of moderately elevated testosterone concentrations in young women. Here we present novel evidence that testosterone alters muscle morphology in these women, resulting in type II fiber hypertrophy and improved capillarization. Our findings suggest that low doses of testosterone potently impact skeletal muscle after only 10 wk. These data provide unique insights into muscle adaptation and support the performance-enhancing role of testosterone in women on the muscle fiber level.

Blood flow restriction (BFR) with low-load resistance exercise (RE) is often used as a surrogate to traditional high-load RE to stimulate muscular adaptations, such as hypertrophy and strength. However, it is not clear whether such adaptations are achieved through similar cellular and molecular processes. We compared changes in muscle function, morphology, and signaling pathways between these differing training protocols. Twenty-one males and females (means ± SD: 24.3 ± 3.1 yr) experienced with resistance training (4.9 ± 2.6 yr) performed 9 wk of resistance training (three times per week) with either high-loads (75%–80% 1RM; HL-RT), or low-loads with BFR (30%–40% 1RM; LL-BFR). Before and after the training intervention, resting muscle biopsies were collected, and quadricep cross-sectional area (CSA), muscular strength, and power were measured. Approximately 5 days following the intervention, the same individuals performed an additional “acute” exercise session under the same conditions, and serial muscle biopsies were collected to assess hypertrophic- and ribosomal-based signaling stimuli. Quadricep CSA increased with both LL-BFR (7.4 ± 4.3%) and HL-RT (4.6 ± 2.9%), with no significant differences between training groups (P = 0.37). Muscular strength also increased in both training groups, but with superior gains in squat 1RM occurring with HL-RT (P < 0.01). Acute phosphorylation of several key proteins involved in hypertrophy signaling pathways, and expression of ribosomal RNA transcription factors occurred to a similar degree with LL-BFR and HL-RT (all P > 0.05 for between-group comparisons). Together, these findings validate low-load resistance training with continuous BFR as an effective alternative to traditional high-load resistance training for increasing muscle hypertrophy in trained individuals. NEW & NOTEWORTHY Low-load resistance exercise with blood flow restriction (LL-BFR) is an effective method for stimulating muscular adaptations, but phenotypical and mechanistic comparisons with traditional high-load training (HL-RT) in trained populations are scarce. The findings indicate that hypertrophy, but not strength, is comparable between LL-BFR and HL-RT, and the acute cellular and molecular processes for hypertrophy were similar, but not identical, between protocols. Thus, LL-BFR is an effective alternative to HL-RT for obtaining hypertrophy in trained populations.

The repair, remodeling, and regeneration of myofibers are dependent on satellite cells (SCs), although, the distribution of SCs in different fiber types of human muscle remains inconclusive. There is also a paucity of research comparing muscle fiber characteristics in a sex‐specific manner. Therefore, the aim of this study was to investigate fiber type‐specific SC content in men and women. Muscle biopsies from vastus lateralis were collected from 64 young (mean age 27 ± 5), moderately trained men (n = 34) and women (n = 30). SCs were identified by Pax7‐staining together with immunofluorescent analyses of fiber type composition, fiber size, and myonuclei content. In a mixed population, comparable number of SCs was associated to type I and type II fibers (0.07 ± 0.02 vs 0.07 ± 0.02 SCs per fiber, respectively). However, unlike men, women displayed a fiber type‐specific distribution, with SC content being lower in type II than type I fibers (P = .041). Sex‐based differences were found specifically for type II fibers, where women displayed lower SC content compared to men (P < .001). In addition, positive correlations (r‐values between 0.36‐0.56) were found between SC content and type I and type II fiber size in men (P = .03 and P < .01, respectively), whereas similar relationships could not be detected in women. Sex‐based differences were also noted for fiber type composition and fiber size, but not for myonuclei content. We hereby provide evidence for sex‐based differences present at the myocellular level, which may have important implications when studying exercise‐ and training‐induced myogenic responses in skeletal muscle.

Age‐related loss of muscle mass and function is underpinned by changes at the myocellular level. However, our understanding of the aged muscle phenotype might be confounded by factors secondary to ageing per se, such as inactivity and adiposity. Here, using healthy, lean, recreationally active, older men, we investigated the impact of ageing on myocellular properties in skeletal muscle. Muscle biopsies were obtained from young men (22 ± 3 years, n = 10) and older men (69 ± 3 years, n = 11) matched for health status, activity level and body mass index. Immunofluorescence was used to assess myofibre composition, morphology (size and shape), capillarization, the content of satellite cells and myonuclei, the spatial relationship between satellite cells and capillaries, denervation and myofibre grouping. Compared with young muscle, aged muscle contained 53% more type I myofibres, in addition to smaller (−32%) and misshapen (3%) type II myofibres (P < 0.05). Aged muscle manifested fewer capillaries (−29%) and satellite cells (−38%) surrounding type II myofibres (P < 0.05); however, the spatial relationship between these two remained intact. The proportion of denervated myofibres was ∼2.6‐fold higher in old than young muscle (P < 0.05). Aged muscle had more grouped type I myofibres (∼18‐fold), primarily driven by increased size of existing groups rather than increased group frequency (P < 0.05). Aged muscle displayed selective deterioration of type II myofibres alongside increased denervation and myofibre grouping. These data are key to understanding the cellular basis of age‐related muscle decline and reveal a pressing need to fine‐tune strategies to preserve type II myofibres and innervation status in ageing populations.

The objective of this work was to investigate myonuclear permanence and transcriptional regulation as mechanisms for cellular muscle memory after strength training in humans. Twelve untrained men and women performed 10 weeks of unilateral elbow‐flexor strength training followed by 16 weeks of de‐training. Thereafter, 10 weeks’ re‐training was conducted with both arms: the previously trained arm and the contralateral untrained control arm. Muscle biopsies were taken from the trained arm before and after both training periods and from the control arm before and after re‐training. Muscle biopsies were analysed for fibre cross‐sectional area (fCSA), myonuclei and global transcriptomics (RNA sequencing). During the first training period, myonuclei increased in type 1 (13 ± 17%) and type 2 (33 ± 23%) fibres together with a 30 ± 43% non‐significant increase in mixed fibre fCSA (P = 0.069). Following de‐training, fCSA decreased in both fibre types, whereas myonuclei were maintained, resulting in 33% higher myonuclear number in previously trained vs. control muscle in type 2 fibres. Furthermore, in the previously trained muscle, three differentially expressed genes (DEGs; EGR1, MYL5 and COL1A1) were observed. Following re‐training, the previously trained muscle showed larger type 2 fCSA compared to the control (P = 0.035). However, delta change in type 2 fCSA was not different between muscles. Gene expression was more dramatically changed in the control arm (1338 DEGs) than in the previously trained arm (822 DEGs). The sustained higher number of myonuclei in the previously trained muscle confirms myonuclear accretion and permanence in humans. Nevertheless, because of the unclear effect on the subsequent hypertrophy with re‐training, the physiological benefit remains to be determined. image Key points: Muscle memory is a cellular mechanism that describes the capacity of skeletal muscle fibres to respond differently to training stimuli if the stimuli have been previously encountered. This study overcomes past methodological limitations related to the choice of muscles and analytical procedures. We show that myonuclear number is increased after strength training and maintained during de‐training. Increased myonuclear number and differentially expressed genes related to muscle performance and development in the previously trained muscle did not translate into a clearly superior responses during re‐training. Because of the unclear effect on the subsequent hypertrophy and muscle strength gain with re‐training, the physiological benefit remains to be determined.