Resistance training has been extensively documented to increase cellular and tissue-level hypertrophy in skeletal muscle (Roberts et al., 2023). Blood flow restriction applied during low-load resistance training (LL-BFR) is an effective strategy to increase the cross-sectional area of muscle fibers (fCSA) (i.e., muscle hypertrophy) (Patterson et al., 2019). Recently, we demonstrated that LL-BFR induces similar hypertrophy of type I and II fibers compared to traditional high-load resistance training (HL-RT), regardless of sex (Reece et al., 2023). Although muscle fiber hypertrophy is similar between LL-BFR and HL-RT, the differential expansion of the myofibrillar (e.g., myosin heavy chain and light chain isoforms, various actin isoforms, z-line proteins, etc.) and non-myofibrillar components (e.g., intracellular fluids, sarcoplasmic proteins and enzymes, glycogen, mitochondria, etc.) may be influenced by these different training protocols. Moreover, no study has aimed to examine if myofiber spacing is differentially affected by these training protocols.

There has been a renewed interest in examining myofibrillar adaptations to resistance training (Jorgenson et al., 2020; Roberts et al., 2020; Roberts et al., 2023). MacDougall et al. (MacDougall et al., 1982) used electron microscopy investigation to show that longer-term resistance training disproportionately increases sarcoplasmic spacing. Contrary to this finding, more recent evidence suggests traditional HL-RT typically induces a proportional expansion in the myofibrillar and non-myofibrillar areas of muscle fibers (Wang et al., 1993; Fox et al., 2021; Ruple et al., 2021; Vann et al., 2022). Additionally, an increase in myofibrillar number, but not size, has been shown to accompanies the increases in fCSA with weeks of resistance training (Jorgenson et al., 2023). However, select evidence suggests that non-myofibrillar area expansion may predominate during muscle fiber hypertrophy in resistance training protocols that diverge from traditional methods (e.g., an unconventional or very high-volume training paradigms) (Haun et al., 2019; Fox et al., 2021; Vann et al., 2022). Despite extensive research into these intracellular changes in recent years, little is known regarding how LL-BFR affects these features in muscle fibers.

To the best of our knowledge, only one study has explored the effects of LL-BFR on intracellular myofibril content. Wilburn et al. (2021) conducted a case study composed by a single exercise session in which one of the subject’s legs underwent LL-BFR, while the contralateral leg was subjected to HL-RT. Vastus lateralis muscle biopsies were obtained before and 30 min after each exercise protocol. The primary findings indicated that the LL-BFR protocol induced acute myofibrillar alterations in a wave-like pattern, likely attributed to the pouches formed by fluid accumulation in the sarcoplasm, which appeared to disrupt the linear alignment of sarcomeres. Additionally, muscle damage (i.e., Z-disc disruption, thick and thin filament rupture) was not observed to the same extent as with HL-RT. These findings suggest that, at least acutely, fluid accumulation induced by LL-BFR may cause changes in the non-myofibrillar area distinct from HL-RT. However, it remains unknown whether these acute changes demonstrated by Wilburn et al. (2021) are also observed a LL-BFR training period.

Therefore, the aim of the present study was to investigate whether the hypertrophy of type I and II fibers induced by LL-BFR or HL-RT is accompanied by differential changes in myofibrillar and non-myofibrillar areas. We additionally examined if extracellular spacing was differentially affected between these two training paradigms. We hypothesized that there would be a decrease in myofibrillar area and an increase in non-myofibrillar area with type I and II fCSA increases following LL-BFR. In contrast, we hypothesized that HL-RT would promote conventional hypertrophy, consistent with findings from previous studies (Fox et al., 2021; Ruple et al., 2021; Vann et al., 2022). Finally, we hypothesized that extracellular area would be greater following LL-BFR.

In this study, we employed histology techniques to investigate whether LL-BFR or HL-RT induces differential changes in the myofibrillar areas of type I and II muscle fibers and extracellular spacing. Contrary to our initial hypothesis, our findings indicate that myofibrillar and non-myofibrillar areas within muscle fibers exhibited proportional expansion in response to an increase in fCSA, independent of training protocols. Additionally, our data revealed that there was no significant change in the extracellular area surrounding muscle fibers. These observations are the first to show that shorter-term LL-BFR and HL-RT promote similar morphological changes in the muscle tissue of previously untrained males and females.

It has been suggested that skeletal muscle hypertrophy induced by mechanical overload can be attributed mainly to the proportional increase in contractile and non-contractile components of myofibers (Jorgenson et al., 2020; Roberts et al., 2023). This phenomenon has been termed conventional hypertrophy (Roberts et al., 2020). In the present study, the HL-RT group showed an increase of ∼17% in fCSA (type I + II). Notably, the myofibrillar area remained at ∼86%, while the non-myofibrillar area remained at ∼14%. These results are consistent with previous studies that estimated the area occupied by myofibrils to be ∼85% of the intracellular space (MacDougall et al., 1982; Alway et al., 1988; Claassen et al., 1989; Ruple et al., 2021). Furthermore, this proportionality appears to be maintained after an HL-RT program performed for ∼10 weeks as reported in prior research (Fox et al., 2021; Ruple et al., 2021).

Conventional hypertrophy, or the proportional increase in myofibrillar area with an increase in fCSA, has recently been challenged by studies that demonstrated sarcoplasmic expansion after some resistance training protocols. For example, Fox et al. (2021) compared traditional HL-RT with a protocol with frequent manipulation of resistance training variables (e.g., load, sets, muscle action, and rest). In contrast to what was observed after HL-RT, the protocol with variation in exercise stimulus induced an increase in the non-myofibrillar area after 16 resistance training sessions. This phenomenon was also demonstrated by Haun et al. (2019) when conducting a study with a very high volume (i.e., number of sets). Taken together, these findings suggest that unconventional training paradigms appear to induce an expansion of non-myofibrillar components (i.e., sarcoplasm). Based on these observations, as well as the acute promotion of fluid accumulation in the sarcoplasm by BFR (Wilburn et al., 2021), our hypothesis was that LL-BFR training would lead to a disproportionate increase in non-myofibrillar area with hypertrophy of type I and II fibers. However, we observed that both protocols led to conventional hypertrophy, and this could be attributed to several possible explanations. First, there is a lack of agreement between acute and chronic responses after LL-BFR. The absence of studies examining myofibrillar and non-myofibrillar components of skeletal muscle using histological techniques makes it difficult to analyze whether persistent intracellular stability after LL-BFR is a prevalent phenomenon. Despite this, some studies have indirectly investigated fluid shifts using ultrasound. In this regard, greater acute cellular swelling was observed when measuring muscle thickness at multiple time points (0 min, 15 min, 60 min, 180 min, and 48 h) following LL-BFR compared to HL-RT (Farup et al., 2015; Freitas et al., 2017). However, these differences were not observed after 16 training sessions (Farup et al., 2015). As the greater early muscle thickness for LL-BFR is unlikely to be primarily due to increased muscle damage induced by this training protocol (Patterson et al., 2019; Wilburn et al., 2021), it is plausible to suggest that the increase in intracellular or extracellular fluid following sessions may subside over time (Reidy et al., 2017). Second, evidence indicates that sarcoplasmic expansion is more frequently observed in well-trained individuals when they engage in an unconventional training regimen compared to novices (MacDougall et al., 1982; Haun et al., 2019; Fox et al., 2021). There is speculation that a threshold for the accumulation of myofibrillar proteins may develop with years of training (Roberts et al., 2020). As a result, muscle fiber hypertrophy might occur through an increase in non-contractile components, strategically preparing muscle cells for the eventual incorporation of myofibrils (Roberts et al., 2020). Hence, the phenomenon of sarcoplasmic expansion following LL-BFR may be a transient occurrence, or one primarily observed in well-trained individuals. Thus, further research is needed to add clarity in this regard.

There are notable limitations to the current study. First, is the limited number of participants. Additionally, phalloidin staining was used for extrapolating myofibril data. Notably, this technique yields a relatively crude two-dimensional rendering of myofiber characteristics and does not take into consideration three-dimensional features that may have been affected (e.g., myofibril branching) (Willingham et al., 2020). It is important to emphasize that we assessed changes in the proportion of myofibril and non-myofibril within muscle fiber cross-sectional area, and not the actual area of each fraction. Moreover, the Hornberger laboratory recently developed an immunofluorescent myofibril imaging technique using deconvolution (termed FIM-ID) that provides exceptional resolution of myofibril number and sizing (Jorgenson et al., 2023). Given that we did not preserve the current tissue accordingly to perform this technique, future examination using a similar study design and said methods are needed. Finally, the limited 18 training sessions warrants further discussion. Indeed, evidence from past shorter-term interventions suggests that 14–21 training sessions is minimally needed to reliably detect myofiber hypertrophy (Goreham et al., 1999; Damas et al., 2016; Mesquita et al., 2023), and this certainly warrants a longer-term intervention with the current training protocols. However, despite the limited training duration, interesting differences between protocols were evident. First, although the CI of ES crossed zero for all variables analyzed, data in Figure 2B show that pre-to-post intervention change scores in type II fCSA between training paradigms presented a moderate effect (ES: 0.73, CI: −0.10–1.70) whereby values were lower in the LL-BFR protocol versus HL-RT protocol (change scores were 19.9% versus 29.3%, respectively). Second, data in Figure 2D show that the pre-to-post intervention change scores in type II myofibrillar spacing between training paradigms presented a moderate effect (ES: 0.77, CI: −0.13–1.66) whereby values were lower in the LL-BFR protocol versus HL-RT protocol (change scores were −2.92% versus 1.63%, respectively). A potential interpretation of these effect sizes is that: 1) longer-term LL-BFR training may be needed to produce comparable type II fiber hypertrophy, and 2) continued type II fiber hypertrophy with LL-BFR may eventually lead to a disproportionate increase in non-myofibrillar spacing. However, given that this speculation is based on moderate effect sizes observed after 18 training sessions with multiple biopsy (acute and chronic) sampling throughout are needed.

In summary, the novel preliminary data demonstrate that 6 weeks of LL-BFR and HL-RT result in proportional expansion in both myofibrillar and non-myofibrillar areas, contributing to the hypertrophy of type II fibers. Given the stated limitations, longer-duration protocols with more advanced imaging techniques (e.g., three-dimensional SEM or FIM-ID) are needed to fully delineate how LL-BRF training affects cellular morphology.