Abstract
ground/Objectives: Acute high-intensity exercise may transiently alter circulating vitamin D metabolites. However, the effects of training status, supplementation dose, and vitamin D-binding protein (VDBP) on the exercise-induced redistribution and bioavailabil- ity of vitamin D remain unclear. This pilot study examined whether training status and vitamin D3supplementation dose modulate vitamin D status, anaerobic performance, and acute post-exercise changes in vitamin D metabolites and VDBP.Methods: Thirty-four healthy young men participated, comprising 22 mixed martial arts (MMA) athletes and 12 untrained controls. MMA athletes received either 3500 or 6000 IU/day of vitamin D3 for four weeks, while untrained participants received 3500 IU/day or a placebo. Before and after the intervention, participants performed a supramaximal anaerobic exercise protocol (3×30 s Wingate tests). Blood samples were collected at rest and 30 min and 24 h post-exercise to assess vitamin D metabolites, VDBP, bioavailable and free 25(OH)D3, interleukin-6, and lactate.Results: Supplementation with 3500 IU/day significantly in- creased serum 25(OH)D3in untrained men (p= 0.003) but not in MMA athletes. In contrast, 6000 IU/day increased 25(OH)D3in MMA athletes to a sufficient concentration (p= 0.001) and improved maximal power (+7.5%), mean power (+4.9%), and total work (+5.0%). Acute exercise increased circulating vitamin D metabolites in trained athletes but reduced
25(OH)D3, interleukin-6, and lactate.Results: Supplementation with 3500 IU/day significantly in- creased serum 25(OH)D3in untrained men (p= 0.003) but not in MMA athletes. In contrast, 6000 IU/day increased 25(OH)D3in MMA athletes to a sufficient concentration (p= 0.001) and improved maximal power (+7.5%), mean power (+4.9%), and total work (+5.0%). Acute exercise increased circulating vitamin D metabolites in trained athletes but reduced them in untrained men supplemented with vitamin D.Conclusions: The efficacy of vitamin D3supplementation and the acute exercise-induced vitamin D responses appear to be training-dependent. A daily dose of 6000 IU is more effective in achieving vitamin D sufficiency and performance benefits in MMA athletes, whereas 3500 IU is sufficient for untrained men. Keywords:vitamin D3; high-intensity exercise; anaerobic performance; vitamin D-binding protein; mixed martial arts 1. Introduction Vitamin D plays a well-established role in calcium–phosphate homeostasis and skele- tal health; however, its relevance in sports medicine has expanded to include skeletal Nutrients2026,18, 1061 https://doi.org/10.3390/nu18071061
Nutrients2026,18, 1061 2 of 19 muscle function, exercise adaptation, and recovery. Athletes training at high intensity, particularly at northern latitudes and during winter months, frequently present with sub- optimal vitamin D status despite increased physiological demands related to repeated muscle contraction and calcium turnover. Consequently, vitamin D3supplementation is commonly recommended in athletic populations; however, optimal dosing strategies and exercise-related responses remain insufficiently defined. Despite the increasing prevalence of vitamin D deficiency in athletes, research investigating the effects of vitamin D3supple- mentation in this population remains limited. Previous studies have shown that vitamin D deficiency may increase the risk of bone injuries such as stress fractures in military and athletic populations [1,2]. Systematic reviews and meta-analyses have also demonstrated that vitamin D3supplementation may improve skeletal muscle strength [3,4]. Recent evidence indicates that acute physical exercise can transiently alter the circulat- ing concentration of vitamin D metabolites. Mosti and coworkers demonstrated that both endurance and strength exercise induce short-term changes in serum 25-hydroxyvitamin D3(25(OH)D3) and related metabolites, suggesting that exercise itself may influence vita- min D3handling rather than acting solely as a passive stressor [5]. Similarly, studies in endurance athletes have shown that prolonged and extreme exercise, such as ultramarathon running, is associated with marked post-exercise increases in circulating vitamin D metabo- lites, indicating the exercise-induced redistribution or mobilization of vitamin D3within the body [6]. However, the magnitude and direction of these responses appear inconsistent across studies and may depend on exercise intensity, duration, baseline vitamin D status, and training level. Importantly, most previous investigations have focused on total circulating 25(OH)D3, with a limited consideration of vitamin D-binding protein (VDBP) and the bioavailable and free fractions that may be more relevant to biological activity. Moreover, little is known about how training status modulates both baseline vitamin D metabolism and the acute response to high-intensity exercise, particularly in strength and combat sport athletes. Other recent research has asked not what vitamin D can do for muscle but what muscle can do for vitamin D. At somewhere between 50 and 100 days [7,8], 25(OH)D3has an unusually long half-life for a steroid hormone, particularly a
modulates both baseline vitamin D metabolism and the acute response to high-intensity exercise, particularly in strength and combat sport athletes. Other recent research has asked not what vitamin D can do for muscle but what muscle can do for vitamin D. At somewhere between 50 and 100 days [7,8], 25(OH)D3has an unusually long half-life for a steroid hormone, particularly a seco-steroid with a broken carbon ring, whereas the half-life of 1,25(OH)2D3is more similar to other steroid hormones, at only a few days. The half-life of VDBP, which transports both the endocrine metabolite and its parent molecule, is also only a few days [9]. These characteristics of 1,25(OH)2D3, alongside its well-known dependence on UVB exposure and thus seasonal variation for vitamin D synthesis, support the existence of an extravascular storage site for vitamin D within the body [10]. Given the lipophilic nature of free vitamin D and that it is purportedly sequestered in the fat mass of obese patients, it is unlikely that the vitamin D in adipose can be mobilized and released as required into the circulation. In cell culture, radiolabeled 25(OH)D3is taken up into mature myotubes but not mature adipocytes [11]. This pilot study focuses on how training status and vitamin D3dose modulate the acute redistribution of vitamin D metabolites and vitamin D-binding protein following a single bout of high-intensity exercise. Therefore, this preliminary study aimed to investigate the effects of two different doses of vitamin D3supplementation on vitamin D status and anaerobic performance in mixed martial arts (MMA) athletes and untrained young men. In addition, we examined the acute response of circulating vitamin D metabolites, VDBP, and vitamin D bioavailability to a single bout of high-intensity anaerobic exercise, with a particular emphasis on differences related to training status. https://doi.org/10.3390/nu18071061
Nutrients2026,18, 1061 3 of 19 2. Materials and Methods 2.1. Study Design This pilot study was conducted as a randomized, double-blind, controlled trial includ- ing two male cohorts: trained mixed martial arts (MMA) athletes and sedentary university students. A total of 34 Caucasian male participants were enrolled (22 MMA athletes and 12 students). Within the athlete group, participants were randomly assigned to receive either a high dose of vitamin D3(6000 IU/day; n = 13) or a lower dose (3500 IU/day; n = 9) for four weeks. The student group was also randomly divided: 6 participants received vitamin D3at 3500 IU/day, and 6 received a placebo in the form of vegetable oil. All interventions were delivered in identical, unmarked containers to ensure blinding. Randomization and supplement allocation were performed by a researcher not involved in data collection or analysis. Randomization was conducted using a computer-generated random sequence, with allocation concealment ensured by an independent researcher. Participants and investigators remained blinded to group assignment until all analyses were completed. Measurements were carried out at two time points: before the intervention period (Before) and immediately after the four-week intervention (After). The study protocol was approved by the Independent Bioethics Committee for Scientific Research at the Medical University of Gda ´nsk (approval No. NKNNB/643/2019–2020) and conducted in accordance with the Declaration of Helsinki. To ensure full methodological transparency and completeness, this study followed the guidelines outlined in the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT). This study was conducted in Gda´nsk, Poland, between September and December 2022, during the late autumn and early winter months, when natural sunlight exposure was limited and endogenous vitamin D synthesis was minimal. Due to the exploratory nature of this study and the limited availability of elite MMA athletes, no a priori sample size calculation was performed. Given the small sample size, the findings should be considered exploratory and interpreted with caution. However, the group sizes were comparable to previous interventional studies conducted in trained MMA populations [12]. This study was prospectively registered at ClinicalTrials.gov (NCT04759729) prior to participant enrollment. Although the initial registration referred specifically to MMA
athletes, no a priori sample size calculation was performed. Given the small sample size, the findings should be considered exploratory and interpreted with caution. However, the group sizes were comparable to previous interventional studies conducted in trained MMA populations [12]. This study was prospectively registered at ClinicalTrials.gov (NCT04759729) prior to participant enrollment. Although the initial registration referred specifically to MMA athletes, the protocol was subsequently extended to include a group of untrained men. Both groups were randomized and received the same supplementation protocol, and the study design and outcome measures remained consistent with the registered trial. The study design is illustrated in Scheme. 2.2. Participants Participants were recruited from Gda´nsk and the surrounding areas. Baseline char- acteristics are presented in Table. Only males aged 18 years or older were included to reduce variability related to sex and developmental stage. The athlete group consisted of individuals with several years of MMA training experience, all of whom had participated in official competitions [12]. Inclusion criteria required that athletes had no diagnosed cardiovascular diseases and no musculoskeletal injuries within the six months preceding this study. Athletes adhered to a standardized MMA training program comprising striking, grappling, strength, and endurance sessions (≥5 sessions/week, 60–90 min per session) and were instructed to maintain their habitual training and dietary practices throughout the intervention. The student group served as a non-training control cohort and did not engage in structured physical activity. This cohort was included to assess baseline vitamin D metabolism and biochemical responses in the absence of regular high-intensity training, rather than as a direct performance-matched control for elite athletes. Written informed https://doi.org/10.3390/nu18071061
Nutrients2026,18, 1061 4 of 19 consent was obtained from all participants after they received comprehensive information regarding the study protocol. Scheme 1.Flow diagram of participants. Table 1.Participant characteristics. Participant Information Untrained- Placebo Untrained-3500 IU MMA-Trained- 3500 IU MMA-Trained- 6000 IU p-Value (Global) Post Hoc p-Value Height (cm) 178.2 ±4.3 176.7 ±6.2 179.3 ±7.7 179.1 ±7.6 0.885 - Weight (kg) 81.5 ±6.9 75.6 ±9.9 80.2 ±9.8 79.4 ±11.9 0.753 - FFM (kg) 67.1±3.0 * c,d 65.0±7.5 71.9 ±8.2 * a 71.1±8.4 * a 0.004 * a vs. c = 0.006 a vs. d = 0.008 FFM (%) 82.7 ±6.0 86.1 ±2.6 89.8 ±2.9 89.9 ±3.7 0.245 - FM (kg) 14.4±5.8 * c,d 10.6±3.1 8.3 ±2.78 * a 8.4±4.3 * a 0.004 * a vs. c = 0.007 a vs. d = 0.008 FM (%) 17.3±6.0 * c,d 13.9±2.7 10.3 ±3.1 * a 10.1±3.7 * a 0.025 * a vs. c = 0.026 a vs. d = 0.036 Values are presented as the mean±SD. The globalp-value refers to the overall comparison among all four groups and was determined using a one-way ANOVA. Tukey’s HSD post hoc test was applied. Values marked with asterisks (*) indicate statistically significant differences (p< 0.05). Superscript letters indicate significant pairwise differences between groups: a —untrained-placebo; c —MMA-trained-3500 IU; d —MMA-trained-6000 IU. FFM, fat-free mass; FM, fat mass. 2.3. Intervention Vitamin D3was supplemented in oil form, with a concentration of 20,000 IU per milliliter (equivalent to 0.5 mg of cholecalciferol), using Miglyol 812 as the excipient. Participants consumed the supplement once daily for four weeks at the assigned dose. Baseline assessments were conducted during the initial visit, while follow-up evaluations occurred the day after the final dose. Participants completed structured interviews before and after the intervention to document health status, medical history, medication use, dietary habits, and training routines. All participants were instructed not to introduce new supplements, alter their habitual diet, or modify physical activity patterns during this https://doi.org/10.3390/nu18071061
health status, medical history, medication use, dietary habits, and training routines. All participants were instructed not to introduce new supplements, alter their habitual diet, or modify physical activity patterns during this https://doi.org/10.3390/nu18071061
Nutrients2026,18, 1061 5 of 19 study. Training load and recovery in athletes were monitored using self-reported session duration and perceived fatigue. 2.4. Body Composition Analysis Pre- and post-intervention body composition was assessed using the Tanita MC-720 segmental analyzer (multifrequency bioelectrical impedance analysis; Tanita Corporation, Tokyo, Japan). Participants stood upright during measurement, avoiding contact between the limbs and torso, while holding hand electrodes and positioning their feet on the device’s sensors. Before each measurement, individuals’ demographic data (age, sex, height) were manually input, and electrodes were cleaned with manufacturer-provided wipes to ensure accuracy. Measurements were conducted under standardized conditions (time of day and nutritional status). Outcomes included body weight, fat mass, and lean mass. 2.5. Blood Sampling and Biochemical Analyses Venous blood samples were collected under standardized conditions at three time points: before exercise (baseline), 30 min post-exercise, and 24 h after the anaerobic test. All participants underwent medical screening before inclusion, and all exercise testing was supervised by qualified personnel to ensure participant safety. Blood was drawn from the arm vein into tubes containing K2EDTA or a coagulation activator. Following centrifugation (3000×g), serum and plasma were aliquoted and stored at−80 ◦ C for further analysis. The serum concentrations of 25(OH)D3, epi-25(OH)D3, 24,25(OH)2D3, and 25(OH)D2 were quantified by employing an isotope dilution technique coupled with liquid chromatography–tandem mass spectrometry (LC–MS/MS). Sample preparation and analy- sis were performed using the Eksigent ExionLC HPLC system equipped with a CTC PAL autosampler (CTC Analytics AG, Zwingen, Switzerland), interfaced with a QTRAP ® 4500 MS/MS detector (Sciex, Framingham, MA, USA). Vitamin D-binding protein (VDBP) and interleukin 6 (IL-6) concentrations were measured using a commercial ELISA kit # K2314, Immundiagnostic, Bensheim, Germany, and 950.035.192, Diaclone SAS, Besancon, France, respectively, according to the manufacturer’s instructions. Samples were processed in dupli- cate, and absorbance was measured at 450 nm using a microplate reader (Varioskan Flash- Spectral Scanning Multimode Microplate Reader 183, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 100µL capillary blood samples were obtained by a trained researcher during both PRE- and POST-intervention testing sessions, collected into sterile graduated microcapillary tubes. Samples were immediately transferred into microcentrifuge tubes
processed in dupli- cate, and absorbance was measured at 450 nm using a microplate reader (Varioskan Flash- Spectral Scanning Multimode Microplate Reader 183, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, 100µL capillary blood samples were obtained by a trained researcher during both PRE- and POST-intervention testing sessions, collected into sterile graduated microcapillary tubes. Samples were immediately transferred into microcentrifuge tubes containing 500µL of 0.6 M perchloric acid and stored at−20 ◦ C until subsequent analysis. Blood collection occurred before the anaerobic exercise test baseline (00) and 15, 30, and 60 min following the anaerobic exercise protocol. Lactate (LA) concentration was de- termined using a colorimetric kinetic assay with a Biosen C-line lactate analyzer (EKF Diagnostic, Barleben, Germany). 2.6. Supramaximal Interval Training Anaerobic performance was assessed before and after the intervention in the morning following a standardized breakfast consisting of an 80 g wheat roll, 60 g strawberry jam, and one banana. Tests were conducted on a cycle ergometer (Monark 884E Sprint Bike, Monark Exercise AB, Vansbro, Sweden), with the saddle adjusted individually for each participant. The protocol began with a 5 min warm-up at 100 watts, during which two brief maximal sprints lasting 3–5 s were performed near the end. After a 3 min rest, participants completed three 30 s all-out supramaximal sprints based on the Wingate anaerobic test, with resistance set to 7.5% of body weight. Two-minute recovery intervals separated each https://doi.org/10.3390/nu18071061
Nutrients2026,18, 1061 6 of 19 sprint. Throughout the sprints, athletes were encouraged verbally to sustain the maximal pedaling cadence. 2.7. Statistical Analysis The complete dataset from 34 participants who fully adhered to the intervention and all study protocols was analyzed. Due to the limited availability of elite MMA athletes, no a priori sample size calculation was performed, and this study was designed as ex- ploratory. The normality of data distribution was assessed using the Shapiro–Wilk test. Descriptive statistics are presented as means with 95% confidence intervals. The baseline characteristics presented in Table (ANOVA) followed by Tukey’s honestly significant difference (HSD) post hoc test when appropriate. A two-way analysis of variance (ANOVA) was used to examine the effects of group and time (as presented in the figures), followed by least significant difference (LSD) post hoc tests where appropriate. For variables assessed repeatedly across multiple time points, time was included as a within-subject factor in the ANOVA model. Baseline serum 25(OH)D concentrations were included as covariates where appropriate to account for inter-individual variability. Statistical significance was set atp< 0.05. All analyses were performed using Statistica software (version 13.3; StatSoft Inc., Tulsa, OK, USA). 3. Results 3.1. The Effect of 4 Weeks of Vitamin D3Supplementation on the Trained and Untrained Young Men Our results showed a different response to the same amount of vitamin D3sup- plementation in MMA-trained and untrained participants. In the untrained men given 3500 IU of vitamin D3for 4 weeks, the baseline 25(OH)D3increased from 30.31±5.34 ng mL −1 before supplementation to 42.11±9.75 ng mL −1 after supple- mentation (p= 0.003), shifting the average into 25(OH)D3sufficiency (>40 ng mL −1 ; FigureA). At the same time, supplementation with 3500 IU of vitamin D 3did not change serum 25(OH)D3concentration in MMA athletes (29.61±3.25 ng mL −1 before, 30.62±3.88 ng mL −1 after). However, the baseline blood concentration in the MMA-trained athletes supplemented with 6000 IU was sufficient to shift the blood 25(OH)D3concentration to a sufficient level, from 30.25±3.24 ng mL −1 before to 42.35±3.37 ng mL −1 after supplementation (p= 0.000). We did not observe a change in the baseline 25(OH)D3concentration in
(29.61±3.25 ng mL −1 before, 30.62±3.88 ng mL −1 after). However, the baseline blood concentration in the MMA-trained athletes supplemented with 6000 IU was sufficient to shift the blood 25(OH)D3concentration to a sufficient level, from 30.25±3.24 ng mL −1 before to 42.35±3.37 ng mL −1 after supplementation (p= 0.000). We did not observe a change in the baseline 25(OH)D3concentration in the untrained-placebo group before and after sup- plementation. Due to the high demand for vitamin D [13], particularly during the winter period in Poland, and the high level of awareness among MMA athletes participating in our study, most of whom routinely take vitamin D3supplements, we did not include a placebo-controlled, MMA-trained group in this study. Our goal was to observe the reaction to two different doses of vitamin D3supplementation in athletes and to examine the blood concentration response of 25(OH)D3to exercise. The untrained men reacted to the dose of 3500 IU; therefore, we did not see a need to supplement this group with a higher dose. Along with the baseline change in 25(OH)D3, we also found a parallel, significant increase in 3-epi-25(OH)D3, reaching a 57% increase in the untrained-3500 IU group (p= 0.015) and a 113% increase in the MMA-6000 group (p= 0.000) and remaining unchanged in the untrained-placebo group and MMA-3500 IU (FigureB). Similarly, 24,25(OH)2D3concentration also significantly rose in the same groups, reaching a 1.49-fold increase in the untrained–3500 IU group (p= 0.003) and 1.69-fold increase in the MMA-6000 group (p= 0.000) and remaining unchanged in the untrained-placebo group and MMA-3500 IU (FigureC). We did not observe any significant changes in 25(OH)D 2concentration (FigureD). https://doi.org/10.3390/nu18071061
Description
The study investigates the effects of vitamin D3 supplementation on MMA athletes and untrained men.