Morphology of proximal and distal human semitendinosus compartments and the effects of distal tendon harvesting for anterior cruciate ligament reconstruction

Abstract The human semitendinosus muscle is characterized by a tendinous inscription separating proximal and distal neuromuscular compartments. As each compartment is innervated by separate nerve branches, potential exists for independent operation and control of compartments. However, the morphology and function of each compartment have not been thoroughly examined in an adult human population. Further, the distal semitendinosus tendon is typically harvested for use in anterior cruciate ligament reconstruction surgery, which induces long‐term morphological changes to the semitendinosus muscle‐tendon unit. It remains unknown if muscle morphological alterations following anterior cruciate ligament reconstruction are uniform between proximal and distal semitendinosus compartments. Here, we performed magnetic resonance imaging on 10 individuals who had undergone anterior cruciate ligament reconstruction involving an ipsilateral distal semitendinosus tendon graft 14 ± 4 months prior, extracting morphological parameters of the whole semitendinosus muscle and each individual compartment from both the (non‐injured) contralateral and surgical legs. In the contralateral leg, volume and length of the proximal compartment were smaller than the distal compartment. No between‐compartment differences in volume or length were found for anterior cruciate ligament reconstructed legs, likely due to greater shortening of the distal compared to the proximal compartment after anterior cruciate ligament reconstruction. The maximal anatomical cross‐sectional area of both compartments was substantially smaller on the anterior cruciate ligament reconstructed leg but did not differ between compartments on either leg. The absolute and relative between‐leg differences in proximal compartment morphology on the anterior cruciate ligament reconstructed leg were strongly correlated with the corresponding between‐leg differences in distal compartment morphological parameters. Specifically, greater between‐leg morphological differences in one compartment were highly correlated with large between‐leg differences in the other compartment, and vice versa for smaller differences. These relationships indicate that despite the heterogeneity in compartment length and volume, compartment atrophy is not independent or random. Further, the tendinous inscription endpoints were generally positioned at the same proximodistal level as the compartment maximal anatomical cross‐sectional areas, providing a wide area over which the tendinous inscription could mechanically interact with compartments. Overall, results suggest the two human semitendinosus compartments are not mechanically independent.


| INTRODUC TI ON
The structure of the musculus semitendinosus (ST) has been of great intrigue in human and comparative anatomy for over 150 years (Humphry, 1869;Macalister, 1868;Parsons, 1898). The ST of many, but not all (Appleton, 1928), species is characterized by the presence of a tendinous inscription (TI) that separates ST into proximal (ST prox ) and distal (ST dist ) neuromuscular compartments, each containing separate nerve innervations (Edgerton et al., 1987;Gans et al., 1989;Hopwood & Butterfield, 1976;Paul, 2001;Roy et al., 1984;Woodley & Mercer, 2005). Although the bicompartmental structure of the human ST has been considered in cadaveric investigations (Barrett, 1962;Garrett et al., 1989;Haberfehlner, Maas, et al., 2016;Kellis et al., 2012;Lee et al., 1988;Markee et al., 1955;van der Made et al., 2015;Wickiewicz et al., 1983;Woodley & Mercer, 2005), in vivo human studies often neglect this unusual design. In vivo studies that have considered both ST compartments have been performed only with typically developing children and those with spasticity Hanssen et al., 2021), while studies with adult participants only quantified TI location with respect to the ischial tuberosity (Kellis et al., 2012;Kellis & Balidou, 2014). Therefore, information on the bicompartmental morphology of ST in living human adults is currently not available.
Besides its peculiar design, ST has functional and clinical importance. Functionally, ST has been recently suggested to be an essential contributor to upright, bipedal locomotion (Tardieu et al., 2022).
Further, sprinters have a relatively large ST (Handsfield et al., 2017), and ST size is correlated with markers of sprint running performance (Takahashi et al., 2021). In orthopedics, the distal ST tendon is routinely harvested as autologous graft tissue, particularly for anterior cruciate ligament reconstruction (ACLR) (Thaunat et al., 2019;Vertullo et al., 2019). Although this surgical procedure essentially sacrifices ST, the ST tendon demonstrates remarkable potential to regenerate and reattach below the knee joint line (Nakamae et al., 2005;Papalia et al., 2015), regaining some level of function.
However, after ACLR, the ST muscle belly is substantially shorter, with decreased anatomical cross-sectional area (ACSA) and volume (Konrath et al., 2016;Makihara et al., 2006;Messer et al., 2020;Morris et al., 2021;Nomura et al., 2015;Snow et al., 2012;Williams et al., 2004). Although the shape of ST, particularly distally, has been qualitatively (Snow et al., 2012) and quantitatively (du Moulin et al., 2023) observed to be different after tendon harvesting for ACLR, it remains unknown if ST prox and ST dist are altered heterogeneously post-ACLR, particularly as other recent work assessing ST regional ACSA at standardized locations of thigh length (Hjaltadóttir et al., 2022) is confounded by ST shortening post-ACLR. As ST prox may function primarily at the hip and ST dist predominantly at the knee (Markee et al., 1955), compartment-specific adaptations, such as shown in musculoskeletal conditions other than ACLR (e.g., children with cerebral palsy; , may be relevant for the common and persistent knee flexion weakness following ACLR with an ST graft, even in the presence of ST tendon regeneration (Konrath et al., 2016;Makihara et al., 2006;Nakamae et al., 2005;Nomura et al., 2015;Papalia et al., 2015).
Overall, knowledge of ST compartment morphology in living human adults and potential (in)congruency in between-compartment adaptions is lacking. Therefore, we used magnetic resonance imaging (MRI) to bilaterally evaluate the morphology of ST, including ST prox and ST dist , in adults post-ACLR with a unilateral distal ST tendon autograft.
Specifically, we aimed to assess whole muscle and compartment morphology on the contralateral (non-surgical) leg, the effects distal ST tendon harvesting has on ST morphology, and how compartments may atrophy with respect to each other, the whole muscle, and the TI. We also aimed to describe the positioning of the TI in relation to compartment and whole muscle morphology and if the positioning may differ on the ACLR leg. We hypothesized the TI would split the ST muscle into two rather homogenous compartments on the non-ACLR leg. Due to distal ST tendon harvesting, we also hypothesized potential betweenleg morphological differences would be greater in ST dist than ST prox .
Participants underwent MRI imaging only after ACLR (424 ± 109 days post-surgery). Single-site ultrasound images were also obtained but are reported elsewhere . All participants, of which five had accompanying meniscal lesions, underwent ACLR with a quadrupled ipsilateral ST autograft (see Section 2.1). and vice versa for smaller differences. These relationships indicate that despite the heterogeneity in compartment length and volume, compartment atrophy is not independent or random. Further, the tendinous inscription endpoints were generally positioned at the same proximodistal level as the compartment maximal anatomical cross-sectional areas, providing a wide area over which the tendinous inscription could mechanically interact with compartments. Overall, results suggest the two human semitendinosus compartments are not mechanically independent.

K E Y W O R D S
graft, hamstrings, magnetic resonance imaging, tendinous inscription, tenotomy Exclusion criteria consisted of: ACLR >6 months after initial injury, concomitant harvesting of gracilis tendon for ACLR, previous major knee injuries, neurological disorders, and/or contraindications for MRI scans. Participants were requested to refrain from strenuous exercise commencing 24 h prior to the investigation and provided written informed consent prior to any involvement in the study. The Griffith University Human Research Ethics Committee (2018/839) approved the study, which was carried out in accordance with the Declaration of Helsinki.

| Surgical procedures
A fellowship trained orthopedic surgeon (CJV) performed all ACLRs. After application of a tourniquet to the thigh, an anteromedial vertical incision was made over the pes anserinus. The sartorius fascia was then incised to visualize the ST tendon. The tendon was left secured to the distal attachment point and an open-ended tendon harvester (Linvatec) was used to release the entire distal tendon length from its muscular attachment. Then, the ST tendon was removed from its distal bony attachment with a scalpel. A quadrupled ST graft was formed using a wrapping technique over two Tightrope fixation devices (Arthrex), proximally and distally, and then sutured using Fibrewire (Arthrex) (Vertullo et al., 2019). The femoral tunnel was created via a transportal drilling technique and the tibial tunnel drilled outside-in. Femoral and tibial fixation with the adjustable fixation devices were undertaken in full extension.

| MRI acquisition and data analyses
With the participant lying supine, T 1 Dixon three-dimensional fast field echo and two-dimensional proton density magnetic resonance images were acquired with a 3T MRI unit (Ingenia; Phillips). Scan acquisition parameters are summarized in Table 1. For T 1 Dixon scans, a B1 field map (dual repetition time) was used to minimize signal contrast variation across the field of view. Coronal T 1 Dixon images were reconstructed into 691 axial slices (1 mm slice thickness) with inplane pixel resolution of 0.446 mm using Mimics software (Version 20.0; Materialise). Each compartment was separately traced in approximately every 5 axial slices in the water in-phase images, with software interpolation used for slices in between. Caution was taken to include as little of the muscle border as possible, and images were manually inspected to ensure interpolation did not cause substantial errors. The ST prox and ST dist masks were also combined and gaps between them filled (i.e., to include the TI, as this is how ST is typically segmented) to create a whole ST mask. Compartment and muscle belly lengths were calculated in the proximodistal axis by multiplying slice thickness (1 mm) by the number of slices in which the respective compartment/muscle was visible (Fukunaga et al., 2001;Messer et al., 2020). The slice containing each compartment's and the entire muscle's largest cross-sectional value was deemed the compartment/muscle maximal ACSA (ACSA max ) (Fukunaga et al., 2001;Kositsky et al., 2020). The location of compartment and muscle ACSA max relative to the respective compartment and entire muscle belly length was also determined, with the proximal end of the muscle corresponding to 0% and the distal end to 100%. Compartment and muscle volumes were calculated by multiplying slice thickness (1 mm) by the sum of contiguous ACSAs (Fukunaga et al., 2001;Messer et al., 2020). The position of the proximal (TI prox ) and distal (TI dist ) endpoints of the TI was determined relative to the length of each compartment and the entire muscle belly, and the proximodistal length (in the axial imaging plane) of the TI was determined from the number of slices in which ST prox and ST dist overlapped. Examples of all morphological analyses are depicted in Figure 1. The distal ST tendon was considered as regenerated if a tendinous structure was visible on proton density and T 1 Dixon scans below the knee joint.

| Statistical analyses
Paired samples t-tests were used to assess the between-leg differences in whole ST muscle morphology (volume, ACSA max , length), while the effects of compartment (ST prox , ST dist ) and leg (contralateral, ACLR) on ST compartment volume, ACSA max , and length were assessed using full-factorial, two-way repeated measures ANOVAs.
These statistical analyses were repeated with the regenerated tendon participant subgroup to confirm grouping all participants in a single cohort regardless of tendon regeneration status did not affect our results. The between-leg differences in TI length and the location of whole ST muscle ACSA max relative to whole muscle and TI lengths were assessed with paired samples t-tests. A two-way TA B L E 1 Acquisition parameters for magnetic resonance imaging scans.

| RE SULTS
Whole ST muscle volume (p < 0.001), ACSA max (p = 0.02), and length (p = 0.001) were all smaller on the ACLR compared to the contralateral leg ( Table 2). The location of whole muscle ACSA max relative to whole muscle length was more distal on the ACLR leg

TA B L E 2
Means and standard deviations of volume, maximal anatomical cross-sectional area (ACSA max ), and length of the whole semitendinosus muscle for contralateral and anterior cruciate ligament reconstructed (ACLR) legs. Paired samples t-tests for betweenleg differences were performed for the entire sample and for the regenerated tendon subgroup but not for the non-regenerated tendon subgroup due to sample size. and TI dist differed only on the ACLR leg (p = 0.025), and not the contralateral leg (p = 0.211), due to a slightly more distal position (relative to muscle length) of TI dist after ACLR (p = 0.007).
The between-leg differences in each morphological parameter (volume, ACSA max , length) were highly correlated between compartments (r ≥ 0.66; p ≤ 0.037; Figure 3). Between-leg differences in compartment volume and ACSA max were strongly correlated with corresponding whole ST muscle differences (r ≥ 0.93; p < 0.001;

| DISCUSS ION
Here, we found ST compartment longitudinal size differs and undergoes compartment-specific adaptions, whereas compartment maximal radial size does not. More specifically, we found volume and length of ST dist were both larger than ST prox in the contralateral leg, while ACSA max did not differ between compartments. In contrast, although both compartments were substantially smaller in the ACLR compared to the contralateral leg, no between-compartment differences in morphological parameters were found in the ACLR leg, suggesting larger volume and length changes in ST dist than ST prox following ACLR. We also found the TI endpoints to generally be positioned around the ACSA max of each compartment. These results provide novel insight into the structure and function of the human ST muscle and how ST compartments have potential to be heterogeneously altered, particularly via their overall lengths.

F I G U R E 4
Pearson's correlation coefficients (r) for the between-leg relative differences in whole semitendinosus (ST) muscle versus proximal (ST prox ; upper) and distal (ST dist ; lower) ST compartment volume (left), maximal anatomical cross-sectional area (ACSA max ; middle), and length (right). Dots represent individual data points from participants with (filled) and without (unfilled) tendon regeneration. All comparisons were significantly correlated (p ≤ 0.013).

F I G U R E 5
Pearson's correlation coefficients (r) for the between-leg relative differences in tendinous inscription (TI) length versus between-leg relative differences in proximal (ST prox ; left) and distal (ST dist ; right) semitendinosus compartment length. Dots represent individual data points from participants with (filled) and without (unfilled) tendon regeneration. Both correlations were significant (p ≤ 0.021).

| ST morphology on the contralateral leg
Here, the volume of ST dist was greater than ST prox on the contralateral (control) leg (Table 3), whereas previous studies Haberfehlner, Maas, et al., 2016;Hanssen et al., 2021) have conflicting results with one another regarding between-compartment volumetric differences, likely due to age and demographic differences between studies (e.g., cadavers, children with or without diseased ST). We also found the proximodistal length of ST dist to be longer than ST prox , which is consistent with studies of cats (Bodine et al., 1982;Edgerton et al., 1987;Loeb et al., 1987) and goats (Gans et al., 1989). The fiber and fascicle length of human ST dist was originally reported to be longer than ST prox (Barrett, 1962;Markee et al., 1955), although more recent dissections suggest average fascicle lengths may be equal between compartments (Haberfehlner, Maas, et al., 2016;Kellis et al., 2012;Wickiewicz et al., 1983;Woodley & Mercer, 2005). However, due to the oblique nature of the TI and muscle-tendon junctions, fascicle length can vary substantially proximodistally (Haberfehlner, Maas, et al., 2016) and depth-wise (Kellis et al., 2012) within a given compartment.
Inferences regarding potential differences in fascicle length from our results are limited as the MRI sequences used only allow for gross morphology to be quantified. Three-dimensional freehand ultrasound Haberfehlner, Maas, et al., 2016;Hanssen et al., 2021) and more complex MRI methods, such as diffusion tensor imaging (Bolsterlee et al., 2019), are needed to quantify compartment fascicle lengths in vivo.
Despite being unable to document at the level of fascicles, the between-compartment differences in length seem to explain the greater volume in ST dist compared to ST prox , as ACSA max did not differ between compartments. ACSA max , a strong determinant of muscle force, and by extension joint torque (Bamman et al., 2000;Fukunaga et al., 2001), is a good proxy of physiological cross-sectional area in muscles with little-to-no pennation, such as ST (Haberfehlner, Maas, et al., 2016;Makihara et al., 2006). Therefore, despite larger volume in ST dist , the lack of a between-compartment difference in ACSA max suggests the maximal force producing capacity of each compartment does not differ in healthy legs. Further, in accordance with previous reports (Lee et al., 1988;van der Made et al., 2015;Woodley & Mercer, 2005), we found the TI originated at approximately one-third of muscle length and continued obliquely into the lower half of the ST, although Garrett et al. (1989) found the TI to terminate slightly more proximally. The TI endpoints (TI prox , TI dist ) were centered approximately in the middle of each compartment, connecting regions where compartments are of their maximal size. Considering forces may be transferred between ST compartments (Bodine et al., 1982;Edgerton et al., 1987;, the TI is well placed to interact between the two compartments, and the possible functional implications of this placement (e.g., force transmission) are discussed below (see Section 4.3).
Practically, TI endpoints coinciding spatially with compartment ACSA max enables the TI to be used as reference to standardize measures of maximal compartment size, which could also be performed using other, more accessible imaging modalities, such as ultrasonography (Haberfehlner, Maas, et al., 2016;Hanssen et al., 2021;Kositsky et al., 2020). However, given the slightly more proximal position of ST dist ACSA max compared to TI dist in the ACLR leg, assessments of ACSA of ST dist after ACLR should include images proximal to the end of the TI, to ensure ACSA max is obtained. Additionally, as the location of whole ST muscle ACSA max along the TI was highly variable, standardized locations for measures of ST ACSA max (e.g., at 50% of TI length; Haberfehlner, Maas, et al., 2016) should be taken with caution as potential inter-limb and/or inter-individual differences at that single location may just be normal variation.

| Effects of ACLR on ST morphology
Between-leg differences in whole ST muscle morphological parameters were comparable with previous studies (Konrath et al., 2016;Makihara et al., 2006;Messer et al., 2020;Nomura et al., 2015;Snow et al., 2012;Williams et al., 2004). Both ST compartments were smaller in volume and shorter in the ACLR leg, but betweencompartment differences in volume and length only for the con- are not independent, even after such drastic morphological adaptations. However, although the two compartments may be mechanically linked, the more substantial shortening of ST dist may reflect a greater change in the length and/or number of sarcomeres inseries (Abrams et al., 2000;Crawford, 1977;Van Dyke et al., 2012).
According to the force-length relationship of muscle, muscle fibers in ST dist may thus be too short to produce high levels of force, particularly at highly flexed knee joint angles corresponding to short ST muscle belly lengths (Wickiewicz et al., 1984). Indeed, experimental results demonstrate substantially decreased knee flexion strength in these highly flexed positions post-ACLR with an ST graft (Makihara et al., 2006;Morris et al., 2021;Nomura et al., 2015). Future studies employing microendoscopy (Pincheira et al., 2022) may be valuable for elucidating compartment-specific changes at the level of the sarcomere across various joint angles post-ACLR.
The non-uniform compartment adaptations post-ACLR are likely explained by the surgical procedure and the effects of epimuscular myofascial connections (Maas & Sandercock, 2010). The proximal ST tendon and muscle portion remain mechanically connected to surrounding tissues and could still contribute to hip and knee joint torques (de Bruin et al., 2011;Maas & Sandercock, 2008). Thus, ST prox is, particularly in the first months following ACLR, likely to experience greater loading than ST dist , which may help maintain its length Wisdom et al., 2015). Further, to harvest the ST tendon, the sartorius fascia is incised and the most distal portion of the ST muscle belly is stripped off the ST tendon with a tendon harvester device. This surgical procedure not only damages the distal muscle end of ST dist but further reduces the myofascial linkages that could maintain some loading through ST dist and physically prevent muscle retraction in the absence of a distal insertion point.
Indeed, attenuated strength deficit and less ST muscle shortening were found when the distal ST insertion was maintained by harvesting only partial tendon width (Sasahara et al., 2014). Future work is needed to determine if this partial ST tendon harvesting technique would also mitigate morphological alterations in ST dist .
The TI was shorter on the ACLR leg, with a near perfect relationship in the between-leg differences in ST prox and TI lengths (r = 0.991; Figure 5). As ST prox fascicles terminate on the TI and a new set of fascicles (i.e., ST dist ) originate from the TI (Barrett, 1962;Garrett et al., 1989;Haberfehlner, Maas, et al., 2016;Markee et al., 1955;Woodley & Mercer, 2005), the distoproximal manner of shortening consequent to distal tendon harvest (Street, 1983) suggests ST dist shortening is unlikely to independently have great influence on TI dimensions, as the TI is proximal to the initiation of shortening. In contrast, distoproximal shortening of ST prox , whose fascicles are distally attached to the TI, is more likely to be the main regulator of TI shortening. Note that although we only measured its proximodistal length, the overall length of the TI must also be shorter in the ACLR leg due to the concomitant radial muscle atrophy. With a smaller radial size, the overall TI length could be equivalent or greater than the contralateral leg only if the TI was oriented more in-parallel with the muscle, which would result in an increased proximodistal length and is not what was found in the present study. The shortening of the TI after ACLR may simply be slackening or crimping as a consequence of geometric constraints or may be plastic modulations, as demonstrated in other aponeuroses after (un)loading conditions (Lee et al., 2006;Wakahara et al., 2015), and should be examined in more detail in future investigations.
Between-leg differences in compartment morphology were still evident in the regenerated tendon subgroup (Table 3), and between-compartment relationships held even when stratified by tendon regeneration status ( Figure 3). However, whole ST muscle ACSA max did not statistically differ between legs in the regenerated tendon subgroup, which may stem from the large within-sample variation (between-leg mean difference: −7.3 ± 14.9%), but may be a result previously overlooked as studies assessing ST ACSA max post-ACLR did not statistically test this parameter in a regenerated participant subgroup against their contralateral or control legs (du Moulin et al., 2023;Konrath et al., 2016;Snow et al., 2012;Williams et al., 2004) or used an average of five 3.6 mm slices when determining ACSA max (Messer et al., 2020). Although unable to statistically compare between subgroups, non-regenerated tendon individuals (n = 3) seemed to have greater between-leg and between-compartment morphological differences (Figures 3-5;   Tables 2 and 3), which is consistent with previous reports of greater shortening and muscle atrophy after a lack of tendon regeneration (Crawford, 1977;Davenport & Ranson, 1930;du Moulin et al., 2023;Konrath et al., 2016;Nomura et al., 2015). While the material and compositional properties possibly differ between regenerated and native tendons (Papalia et al., 2015), the more substantial morpho-

| Role and function of the TI
The placement of an oblique, full-thickness TI within the human (and a variety of mammalian) ST has been puzzling anatomists for over a century (Humphry, 1869;Parsons, 1898). Ontogenetically, it is thought the TI marks the fusion between two separately developing anlagen (Bardeen, 1906;Macalister, 1868) and is possibly a neomorph (Appleton, 1928) resulting from the crossing of two muscles (Haines, 1934). However, Parsons (1898) noted a TI is not always present at the union of two muscle heads and thus there may be further morphogenetic explanations. Parsons' sentiments were supported by later works finding a small number of fascicles bridge the TI and course from ST prox to distal tendon insertion (Loeb et al., 1987;Markee et al., 1955;Woodley & Mercer, 2005), and that a TI separating compartments can also be present even when ST prox is itself divided into two (dorsal and ventral) heads . As fascicles generally terminate (ST prox ) or originate (ST dist ) on the TI and to date intrafascicularly terminating muscle fibers within the human ST have not been observed (Barrett, 1962;Woodley & Mercer, 2005), the TI potentially serves to simply connect in-series muscle fibers (Humphry, 1872;Trotter et al., 1995). Connecting serial muscle fibers through a TI would allow fibers to be of various lengths and experience varying levels of strain (Loeb et al., 1987), potentially reducing the risk of fiber damage without severely affecting ST function, given its wide joint-level operating range (Cutts, 1989;Peters & Rick, 1977), and allow for deep-to-superficial subunits within a given compartment (Bodine et al., 1982;Chanaud et al., 1991;Kellis et al., 2012). However, intrafascicularly terminating muscle fibers have been found in other human muscles with TIs (e.g., rectus abdominis; Cullen & Brödel, 1937;Woodley et al., 2007) and within ST compartments in other mammals whose ST contains a TI (Gans et al., 1989;Loeb et al., 1987). Therefore, even if human ST fibers do span entire fascicles, the TI seems to have another, main functional role than to just connect serial fibers.
Using shear-wave elastography, we recently indirectly demonstrated passive forces do not differ between human ST compartments, although it was unclear if forces were independently but equally developed or transmitted from one compartment to the other, resulting in equilibrium across the whole muscle .
These findings corroborate data from cat ST, whereby forces appear to be transferred between compartments (Bodine et al., 1982;Edgerton et al., 1987). Here, we document the TI is advantageously positioned to possibly assist in force transmission by connecting the largest regions of each compartment (Figure 2), and this placement generally remains after the substantial gross morphological changes induced by harvesting the ST tendon for ACLR. As efficient force transmission from muscle fibers to the connective tissue network occurs through shear at fiber ends (Purslow, 2020), the oblique arrangement of the TI provides a geometrical design facilitating shearing at the junction between fiber and connective tissue that would not be possible if the TI was completely transverse or coursing in the fascicle direction. The consequence of such an anatomical arrangement could allow for redistribution and transmission of forces across fascicles of each compartment, as suggested by Kellis et al. (2012). In support of a force transmission role of the TI, muscle fiber-TI connections have been reported to be comparable with myotendinous junctions (Hijikata & Ishikawa, 1997), and the TI of other muscles, such as in the cat neck, has been shown to house and/or be surrounded by Golgi tendon organs and muscle spindles (Richmond & Abrahams, 1975a, 1975b. Should the TI of ST also contain these sensory receptors, detection of local forces by Golgi tendon organs (Maas et al., 2022) and muscle spindles (Smilde et al., 2016) combined with the potential for asynchronous activation (English & Weeks, 1987;Hutchison et al., 1989) and unequal strains (Edgerton et al., 1987;Markee et al., 1955) between compartments provides a mechanism by which the central nervous system could use the TI to regulate compartmental force and stiffness to control intercompartmental coordination and enable efficient force transmission between compartments. Future studies combining complex computational models assessing force transmission (Sharafi & Blemker, 2011;Zhang & Gao, 2012) and muscle fiber interaction with internal aponeuroses (Knaus et al., 2022) may be able to clarify the main functional role(s) of the TI.

| Limitations
We only used the contralateral, non-surgical leg as the control measure. However, unlike the quadriceps, hamstring morphology on the injured leg remains unchanged following anterior cruciate ligament injury alone (Kariya et al., 1989;Konishi et al., 2012;Lorentzon et al., 1989;Williams et al., 2004), and after ACLR the morphology of ST on the non-injured leg does not differ compared to pre-surgical (Williams et al., 2004) and control (du Moulin et al., 2023; Morris et al., 2021) groups. Additionally, the substantial between-leg differences in morphology found here compare well with previous literature (Konrath et al., 2016;Makihara et al., 2006;Messer et al., 2020;Nomura et al., 2015;Williams et al., 2004) and exceed bilateral asymmetry measures previously reported for ST (Kulas et al., 2018;Speedtsberg et al., 2022;Williams et al., 2004).
Therefore, using the contralateral leg as the baseline control in a sample of 10 participants was unlikely to have influenced the results.
Further, compartment length was quantified by the proximodistal length of the respective compartment. As the TI is a complex threedimensional structure, compartments are comprised of fascicles of various lengths (Haberfehlner, Maas, et al., 2016;Kellis et al., 2012) and thus compartment length may not accurately represent fiber or fascicle length. Therefore, we do not make any concrete conclusions at length scales below proximodistal compartment length as they were not possible to assess from our MRI scans. Finally, the ACLR surgical intervention induces secondary trauma at the knee joint and is thus more complex than regular tenotomy. However, slightly greater changes in ACSA are seen in the distal compared to proximal gracilis muscle when its distal tendon is harvested for shoulder reconstruction (Flies et al., 2020). Therefore, the results found in the present study are likely due to the ST tendon harvest for the ACLR procedure rather than post-ACLR immobilization and disuse, but should be confirmed in future studies assessing ST compartment alterations after an ST tendon autograft has been used for reconstructing other lower (Cody et al., 2018;Stenroos & Brinck, 2020) and upper (Ranne et al., 2020;Virtanen et al., 2014) limb tendons. The compartment alterations in the ACLR leg may also not be representative of other (un)loading conditions, whose adaptations can also be assessed using the MRI acquisition parameters presented in this study.

| CON CLUS IONS
The proximal and distal compartments of the human ST muscle appear to be modified in a non-uniform manner following harvest for ACLR. However, the heterogenous changes in length do not affect the homogeneity in compartment maximal radial size. The location of the TI with respect to compartment morphology provides a wide area over which this connective tissue sheath could mediate the mechanical interaction of ST compartments. Overall, these results suggest the proximal and distal compartments of the human ST muscle are not mechanically independent.