An initial survey was conducted to gather demographic details, including the participants' age, sex, ethnicity, height, weight, years of playing experience, and position. Evaluation of current AT pain and functionality was conducted using the Victorian Institute of Sport Assessment-Achilles (VISA-A). This questionnaire serves as a reliable assessment tool for the AT (44). Participants then completed a detailed questionnaire assessing their lower limb injury history (Hx). The questionnaire instructed them to report all lower-body injuries they had sustained that had prevented them from participating in their sport in the 12 months preceding the completion of the questionnaire. The players were also required to indicate the injury location, duration, and whether the injury was recurrent or isolated.

Participants underwent US evaluation to assess thickness, EI, fibrillar pattern, and resting stiffness of the AT using Concordia University School of Health's Aixplorer US unit (Supersonic Image, Aix-en-Provence, France; software version V12.2.0.808) equipped with a high-frequency linear array transducer (SL 15–4, 4–15 MHz). Tendon stiffness was primarily quantified as Young's modulus (kPa) as provided by the Aixplorer SWE software. Shear wave speed (SWS) (m/s) was additionally recorded for descriptive purposes, but all subsequent analyses were performed using Young's modulus. US imaging and SWE assessments were performed in accordance with international musculoskeletal US guidelines published by the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB) (45, 46). US examinations and measurements were performed by a single trained sonographer (master's-level researcher) who completed multiple supervised training sessions with experienced musculoskeletal sonographers and conducted test–retest assessments on randomly selected participants to ensure measurement reliability. The system was operated using a standardized Musculoskeletal—Foot and Ankle preset, with imaging depth set to 2.0 cm and the SWE full-scale range set to 600 kPa to avoid signal saturation. Overall gain and image-processing parameters were optimized at the beginning of data collection and maintained constant across all participants, and the focal zone was consistently positioned at the level of the AT. All measurements were taken between October and December 2024 in the same room at Concordia University's School of Health, with room temperature regulated at 21 degrees Celsius.

The players were instructed not to engage in any form of training on the day of assessment before the measurements to ensure reliable and precise AT stiffness measurements (35, 47). Shoes and socks were removed to allow for the probe's direct access to the skin. Players were then instructed to lie prone with their feet hanging off the edge of the examination table in a relaxed position to ensure more reliable results (29). Gel was applied to the targeted area, and the high-frequency linear probe was aligned along the axis of the AT. Consistent light pressure was maintained during the measurements, with both the evaluator and the participant minimizing their movements. AT was identified and pictured in B-mode by placing the transducer on the posterior aspect of the lower limb, 3 cm proximal to the tendon's insertion onto the calcaneus. At this location, three images were captured on each side for the analysis of tendon thickness, EI, and fibrillar pattern. This site was chosen due to its anatomical relevance, avoidance of saturation, and reproducibility reasons. The tendon's mid portion (2–6 cm from the calcaneus) is considered the avascular zone of the tendon. It is commonly affected in tendinopathy and is most relevant to clinical pathology, as it presents significant variations in SWE readings when affected (28). The distal tendon shows very high SWE measurements, and in athletes who already have elevated AT stiffness, measurements taken closer to the calcaneus may approach or even exceed the Aixplorer's maximum detectable limit (16.3 m/s) (4). Saturation and reduced measurement accuracy are avoided by taking measurements at the midportion. After the target region was identified and imaged, the evaluator switched to SWE mode, allowed a brief stabilization period (approximately 3–5 s) to ensure signal stability, froze the image, and proceeded to obtain SWE measurements using a constant round ROI of 3 mm in diameter (Figure 1). The constant 3 mm circular ROIs are in accordance with previous research (36, 40), and were used to standardize sampling. These were positioned within the mid-portion of the tendon core to avoid tendon borders and visible artefacts or heterogeneous regions on the elastography map. Three measurements were obtained per side and averaged to reduce pixel-level variability. The mean was calculated and used in the analysis.

The analysis of AT thickness, EI, and fibrillar pattern was conducted retrospectively using the captured images. Thickness and EI were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Thickness was determined as the mean of the thickest segment measured across the three images, in accordance with previously established methodologies (48, 49). For each longitudinal B-mode image, tendon thickness was measured in ImageJ as the maximum anteroposterior distance between the superficial and deep tendon borders, using a perpendicular caliper placed at the visually identified thickest point along the imaged tendon segment. EI was calculated as the mean grayscale value of the manually traced tendon across the three images; this value for a pixel could range from 0 (dark) to 255 (bright), adapted from previously employed methodologies (50). Fibrillar pattern was evaluated visually for each tendon by the investigator and categorized as one of either fine, parallel, and linear fibrillar pattern or disrupted, fragmented, and absent fibrillar pattern, adapted from established categorization techniques (24, 51).

Participants' dominant jumping side was determined by their single-leg vertical jump test results, with the better-performing side identified as the DJL.

The distance-to-wall technique was used in a weight-bearing lunge position to measure ankle joint dorsiflexion. Previous studies have found that this technique is more accurate than non-weight-bearing methods in assessing ROM (59). It is cost-effective and eliminates the need for specialized equipment, which makes it suitable for reliable execution by novice evaluators (59). Participants were instructed to touch the wall with two fingers from each hand to maintain stability while performing a forward lunge. With the test foot placed perpendicular to the wall, participants lunged forward until their front knee made gentle contact with the wall while ensuring the front foot remained flat on the ground. Subsequently, the testing foot was gradually shifted away from the wall until the knee barely touched it. Ankle dorsiflexion ROM measurement was identified as the maximum distance between the big toe and the wall while maintaining knee-wall contact and without lifting the heel.

Our findings indicate that males exhibit greater AT thickness and a tendency toward increased stiffness compared with females, although the latter did not reach statistical significance. These observations are consistent with prior reports in the literature (11, 49, 61–63). Such differences likely reflect inherent physiological variations between sexes, particularly in muscle mass and hormonal profiles, which influence AT properties (2, 62, 64). Tendons adapt to mechanical loading by increasing stiffness and thickness to accommodate the forces transmitted through the muscle-tendon unit (64). Since males generally exhibit greater body mass and force production (65), the resulting mechanical stress likely drives these adaptive changes.

Reduced AT cross-sectional compliance, which is a measure of the tendon's deformation across its thickness when loaded, may also explain the increased stiffness observed in males (61). Male tendons exhibit less elongation under equivalent loading, which indicates a diminished capacity for deformation and thus greater resistance to stretch (61, 62). In contrast, females typically demonstrate higher AT compliance, which may enhance adaptability to mechanical loading and is associated with lower tendon stiffness (61). Females also display elevated estrogen levels, which have been linked to altered collagen synthesis and show a negative association with tendon stiffness (62, 66).

While structural and mechanical sex differences in the AT are increasingly well-documented, imaging characteristics such as EI remain less explored. To date, no study has specifically examined sex differences in AT EI among athletes. Previous research, however, has reported lower EI values in the gastrocnemius and quadriceps of males compared with females (67). These differences are likely attributable to the higher proportion of lean muscle mass in males, characterized by a greater content of contractile tissue and water, which manifests as darker ultrasonographic images (67).

Consistent with previous research, our findings suggest that AT stiffness is not influenced by leg dominance (2, 22). While no studies to date have examined sagittal-plane differences in AT thickness between the DJL and NDJL in basketball players, prior investigations provide relevant insights. Specifically, no significant differences in AT thickness were observed between the right and left limbs of semi-professional running athletes (6), nor between the dominant and non-dominant sides in healthy young adults when assessed in the transverse plane (68). Similarly, to our knowledge, no studies have evaluated EI asymmetries between dominant and non-dominant ATs in performance-based athletes.

Although a previous study on university-level basketball players reported a significant increase in jump height of the dominant side over the other (21), our results indicate that the performance asymmetry is not associated with differences in AT stiffness, thickness or EI. Instead, these discrepancies in performance may be explained by ankle joint kinematics. Specifically, the dominant leg has demonstrated shorter ground contact times, larger ankle joint angles, and greater angular velocities than the non-dominant leg (21). While jump performance between the two legs was not associated with any of the US derived properties, 35% (n = 11) of the players demonstrated a jump height asymmetry greater than 10%, which has been argued to increase the risk of injury (21). This suggests that neuromuscular or biomechanical factors beyond tendon structure likely underlie functional asymmetries.

It is also important to note that our study employed a different definition of leg dominance than certain previous studies (2, 18, 22). The DJL was defined as the leg that produced the highest running single-leg jump height. This objective, performance-based criterion was selected because basketball players frequently display unilateral jump asymmetries and often struggle to identify their dominant leg subjectively. By reducing subjectivity, this approach provided a more standardized method for distinguishing the DJL from the NDJL.

Our findings suggest that a recent Hx of injury may be associated with differences in AT stiffness in university-level athletes. Reported injuries affected the ankle, knee, hip, and shins, with ankle and foot injuries being the most prevalent (58% of all cases). Previously injured male players exhibited reduced stiffness compared to their non-injured counterparts. However, these findings should be interpreted with caution due to the relatively small sample sizes within some injury subgroups, particularly among female athletes. These results point toward a potentially complex and sex-specific relationship between AT stiffness and lower body injury, but do not allow for definitive conclusions. On the contrary, no differences were found between the previously injured group and the non-injured group in AT thickness or EI.

Numerous studies have examined the direct relationship between US properties and AT injuries, focusing primarily on tendinopathy and tendon rupture injuries. These have consistently reported a significant reduction in stiffness and EI, as well as an increase in thickness associated with both conditions (36, 69–71). Our results extend this evidence by suggesting that broader lower-limb injury Hx, not limited to tendon-specific pathology, may also be associated with alterations in AT stiffness. However, the inclusion of heterogeneous injury types and limited subgroup sizes may partially explain the absence of associations between AT thickness or EI and injury Hx.

These observations warrant further investigation using larger sample sizes within sex- and injury-specific subgroups to obtain more comprehensive assessments of the relationship between injury and AT properties. Future studies could, for example, track SWE, thickness, and EI measurements longitudinally across an athletic season, throughout recovery phases, and following bouts of exercise.

Tendons with disrupted fibrillar architecture, which we defined as fragmentation, interruption, or loss of the normal linear fibrillar pattern, showed no differences in US-derived properties or in objective measures of functional performance in comparison to ATs with normal, organized fibrillar patterns. While most research has focused on the fibrillar pattern after AT reconstructive surgery, to our knowledge, no prior studies have evaluated this property as a determinant of structural characteristics and functional outcomes in athletic populations.

Emerging imaging evidence offers relevant insights. A recent study investigating tendon structure in healthy and symptomatic cohorts demonstrated that six weeks of ballet training induced an increase in fibrillar disorganisation without associated changes in clinical symptoms or functional performance (51). In contrast, longitudinal imaging in elite football players showed that the fibrillar pattern of asymptomatic AT's improved across a 5-month pre-season, with increases in aligned fibrils and decreases in disorganized echo-types (72). Together, these findings suggest that AT fibrillar pattern is dynamic and responsive to load but may not directly determine functional outcomes or other US-derived properties.

Within the US-derived measures assessed, only thickness and EI demonstrated a consistent association. In both males and females, this relationship was characterized by a strong negative correlation, suggesting that increased thickness may be associated with reduced EI. A potential explanation is that greater thickness reflects pathological changes, such as fiber disorganization and elevated interstitial fluid, which contribute to decreased EI.

The current evidence supporting this association remains inconclusive. For instance, in asymptomatic pre-professional dancers, AT thickness did not differ between groups with distinct EI patterns (25). In contrast, a study in badminton players, which included both tendinopathic and healthy tendons, reported greater stiffness in ATs with more heterogeneous echogenicity, which the authors suggested could represent a transition from a physiological to a pathological state (73).

Overall, these findings indicate that the relationship between thickness and EI is context-dependent, with stronger associations observed in the presence of disease or injury.

Although mean EI provides a well-established and reproducible measure of tendon echogenicity, it represents a global summary of grayscale values and does not capture spatial heterogeneity within the tissue. Advanced texture-based US metrics, such as entropy, contrast, or correlation, may offer additional insight into tendon microstructural organization by quantifying grayscale variability and spatial patterns. The inclusion of such texture analyses was beyond the scope of the present study but represents an important avenue for future research aimed at further characterizing tendon structural adaptations in athletic populations.

Unlike prior work, our findings did not reveal a significant correlation between AT stiffness and ankle ROM (19). A recent study using SWE and a digital goniometer-based lunge test in 20 healthy young individuals reported a moderate negative correlation between ankle dorsiflexion ROM and AT stiffness (19). Several methodological differences may explain the discrepancy with our results: the participants involved in their study were non-athletes, their ROM testing procedures differed from our knee-to-wall test, and they only performed SWE on the right limb of the participants (19). These discrepancies make direct comparison less reliable.

Although the AT plays a role in ankle ROM based on its anatomical insertion, several other factors also influence ankle ROM, including joint capsule stiffness, sex, age, pain and stretch tolerance, Hx of ankle injury, and the athlete's warm-up state (18, 74–76). These influences likely dilute the role of AT stiffness, helping explain the absence of significant correlations in our cohort.

Support for this interpretation comes from related tendon and muscle studies. Gastrocnemius EI has shown no correlation with maximal dorsiflexion ROM (74), and AT thickness likewise shows no association with ROM in young collegiate athletes (77). Taken together, these findings suggest that structural properties of the tendon and muscle alone may not be sufficient to explain individual differences in ankle dorsiflexion. Instead, ankle ROM should be understood as an emergent property shaped by a broader set of biomechanical and neuromuscular factors.

Previous studies have demonstrated that greater functional stiffness in the lower limb can enhance performance (3, 14, 15). However, in our cohort, local AT stiffness, thickness, and EI did not correlate with jump height. Although the type of jump used in our study differed from those in earlier reports, our findings are consistent with two previous studies that reported no correlation between AT stiffness and jump height (78, 79). These studies analyzed the tendons' behaviour during physical tasks to deduce a measure of functional tendon stiffness, rather than SWE, and employed slightly different jump test protocols, yet yielded similar results (78, 79).

The lack of association is likely explained by the multifactorial nature of jump height (80). Age, sex, body composition, power output, and jumping technique all influence jump performance (81–84). While tendon properties may contribute, their relative effect is likely outweighed by these other variables. In particular, EI appears to have little predictive value for functional outcomes in healthy or athletic populations (25). Therefore, these US-derived properties may not be major determinants of jump height, which is a complex task requiring contributions from multiple muscle groups and a high degree of coordination.

A novel aspect of our study was the exploration of correlations between AT properties and HR test outcomes (repetition count and total HR height). It was initially hypothesized that these outcome measures would reflect the strength of the ankle plantar flexor muscle-tendon unit and correlate with AT US-derived health indicators (85). However, our findings did not support this assumption. One explanation is that the HR test may be a more accurate indicator of muscular endurance than strength. Endurance performance is shaped by a range of physiological and neuromuscular factors, including body composition, training Hx, oxygen uptake, blood flow, capillary density, mitochondrial capacity, and preferential recruitment of type I fibers (86–90). All of which were not assessed and may have influenced the results.