Extended Abstract
1.    Introduction
Dietary supplements—defined as nutrients or bioactive compounds used to enhance health or performance beyond what is typically achieved through a standard diet—are widely consumed by both athletes and the general population (1–3). Athletes commonly use supplements not only for direct performance enhancement but also for indirect benefits such as supporting training adaptations, optimizing body composition, facilitating injury recovery, and improving mood and well-being. Over the past two decades, supplement use has increased substantially and now includes a broad range of products, including carbohydrates, proteins, vitamins, minerals, herbs, and plant-derived extracts (1,4–12). Vitamin D, a secosteroid prohormone and precursor of the active metabolite calcitriol, plays a central role in calcium–phosphate homeostasis, bone metabolism, skeletal muscle function, and the modulation of inflammatory responses. It has been shown to suppress pro-inflammatory cytokines such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), while upregulating the anti-inflammatory cytokine interleukin-10 (IL-10). In addition, vitamin D promotes cytoprotective effects in tenocytes through activation of ERK and p38 MAPK signaling pathways, mechanisms that may contribute to tendon repair and tissue homeostasis (13–18). Previous evidence suggests that vitamin D supplementation may enhance tendon strength, stimulate collagen synthesis, and support extracellular matrix integrity, particularly in populations exposed to high mechanical loads, such as athletes, or in aging individuals, potentially accelerating recovery following tendon injury (19–22).
The biomechanical properties of tendons—including maximum force (Fmax), stiffness, deformation, and absorbed energy—are key indicators of their functional capacity and resistance to mechanical loading. Maximum force reflects ultimate tensile strength, stiffness represents resistance to deformation, deformation characterizes elastic behavior, and absorbed energy indicates the tendon’s ability to withstand and dissipate mechanical energy before failure (23,24). These properties are influenced by tendon morphology, collagen organization, extracellular matrix composition, and molecular and cellular factors governing tissue remodeling (25–28). Despite the well-established role of vitamin D in musculoskeletal health, its specific effects on tendon biomechanics remain insufficiently investigated, particularly beyond stiffness and across different experimental models. Therefore, the present study aimed to evaluate the effects of an eight-week vitamin D supplementation protocol on the biomechanical properties of the Achilles tendon in male Wistar rats. It was hypothesized that vitamin D supplementation would lead to improvements in tendon stiffness, tensile strength, and overall mechanical resilience.
2.    Methods
In this experimental study, 30 male Wistar rats (aged 2–3 months, initial body mass 180–230 g) obtained from the Royan Institute (Tehran, Iran) were housed at Qom University of Medical Sciences. Animals were acclimatized for one week in polycarbonate cages under controlled environmental conditions (22 °C, 12:12 h light–dark cycle) with ad libitum access to standard chow and water. Rats were randomly allocated into three groups (n = 10 per group): control, paraffin (vehicle), and vitamin D₃ supplementation.
The vitamin D₃ group received 500 IU/kg body weight (equivalent to 12.5 μg/kg), dissolved in paraffin oil (1.5 mg/kg), administered via oral gavage three times per week for eight weeks (29,30). The paraffin group received the vehicle only, while the control group received no intervention. All experimental procedures were conducted in accordance with the ethical guidelines for the care and use of laboratory animals at Qom University of Medical Sciences and were approved by the Ethics Committee of the Physical Education and Sport Sciences Research Institute (approval code: IR.SSRI.REC.2312.2570).
Twenty-four hours after completion of the intervention period, rats were anesthetized using ketamine (100 mg/kg) and xylazine (80 mg/kg). The Achilles tendons were carefully excised, placed in Falcon tubes to prevent dehydration, and subjected to biomechanical testing. Tendon mechanical properties were assessed using a Santam® tensile testing machine, and stress–strain curves along with rupture loads were recorded using the associated software. Descriptive statistics were calculated and are reported as means and standard deviations. Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was evaluated with Levene’s test. Between-group comparisons were performed using one-way analysis of variance (ANOVA), with the level of statistical significance set at p < 0.05. Statistical analyses were conducted using SPSS software.
3.    Results
The Shapiro–Wilk test confirmed normal data distribution, and Levene’s test indicated homogeneity of variances, thereby satisfying the assumptions for ANOVA. One-way ANOVA revealed no significant effects of vitamin D supplementation on Achilles tendon Fmax, stiffness, absorbed energy, or deformation (p > 0.05; Tables 1 and 2). 

 

4.    Discussion
In the present study, mean maximum force (Fmax) values did not differ significantly between groups, indicating that vitamin D supplementation does not enhance ultimate tendon strength in healthy animals. This finding is consistent with earlier work by Simonsen et al. (1995) and Sommer (1987), who demonstrated that mechanical loading is the primary determinant of tendon strength once nutritional requirements are met, with dietary factors exerting only a limited influence under normal conditions (31,32). In this context, vitamin D appears to act in a permissive rather than an augmentative manner. In contrast, Angeline et al. (2014) reported reduced Fmax and impaired healing in vitamin D–deficient rotator cuff models, while Min et al. (2019) observed restored collagen type I synthesis and increased tenocyte proliferation in vitro following vitamin D exposure (17,33). These discrepancies are likely attributable to differences in baseline vitamin D status, the presence of tissue injury, and experimental design, supporting the notion that supplementation does not confer additional tensile strength benefits in the absence of deficiency or structural damage.
Similarly, no significant changes in Achilles tendon stiffness were observed following vitamin D supplementation. The slight, non-significant reduction in stiffness in the vitamin D group may reflect minimal biological variability, such as subtle changes in extracellular matrix hydration or collagen cross-linking, rather than true mechanical adaptation. These findings align with those of Kubo et al. (2001), who reported no stiffness alterations following short-term interventions in the absence of mechanical loading (34). Conversely, studies by Tarantino et al. (2024) and Dougherty et al. (2016) demonstrated increased collagen content and reduced matrix metalloproteinase-9 (MMP-9) expression in vitamin D–deficient conditions (20,35), suggesting that supplementation alone is insufficient to induce measurable biomechanical remodeling in healthy tendon tissue.
Although the vitamin D group exhibited the highest mean deformation values, these differences did not reach statistical significance. This trend may indicate a slight increase in tendon compliance or viscoelastic behavior; however, given the absence of concomitant changes in Fmax or stiffness, it is more likely attributable to normal biological variability rather than a meaningful adaptive response. Huang et al. (2004) similarly reported non-significant changes in deformation following mild interventions in healthy tendon models (24). In contrast, Guan et al. (2022) observed reduced deformation in osteoporotic models following vitamin D supplementation (21), further supporting the hypothesis that vitamin D primarily exerts protective or restorative effects in compromised tissues rather than enhancing mechanical properties in healthy tendons.
Absorbed energy, a measure of tendon toughness, also did not differ significantly among groups. This finding is consistent with previous animal studies demonstrating stable energy absorption in the absence of prolonged overload, injury, or pathological conditions (32). While Min et al. (2019) and Kim et al. (2022) reported that vitamin D can enhance extracellular matrix integrity at the cellular level (17,18), studies by Huang et al. (2004) and Sommer (1987) emphasized that changes in absorbed energy are more strongly influenced by mechanical loading magnitude and rate than by nutritional supplementation in healthy animals (24,31). It is therefore plausible that molecular or histological alterations induced by vitamin D require longer intervention periods or the presence of injury or deficiency to translate into detectable biomechanical changes. Overall, the absence of significant effects across all measured biomechanical parameters—Fmax, stiffness, deformation, and absorbed energy—suggests that vitamin D supplementation does not provide additive mechanical benefits to structurally intact and physiologically sufficient tendons. These findings underscore the context-dependent nature of vitamin D’s role in tendon biomechanics, with its influence becoming more apparent under conditions of deficiency, inflammation, or tissue damage, where it modulates collagen synthesis, cellular proliferation, and inflammatory pathways (20,35). In contrast, in healthy tendons, structural and functional properties may already operate near an optimal plateau, beyond which additional vitamin D supplementation yields no measurable biomechanical enhancement. 

Ethical Considerations
Compliance with ethical guidelines
This study was conducted in compliance with the standards of research ethics and the protocol of this study was approved by the Ethics Committee of the Institute of Physical Education and Sport Sciences with the ethics code IR.SSRI.REC-2312-2570.
Funding
The authors have not received any financial support from any government or private organization or institution. 
Authors' contributions
All authors contributed equally to preparing the article.
Conflicts of interest
The authors declare that they have no conflict of interest associated with this study.