The Urban Central Region Institutional Review Board at Intermountain Healthcare (Salt Lake City, UT, USA) approved this study. Subjects were informed of and provided written and verbal consent to the experimental protocol and procedures. Reportedly healthy and modestly-active (i.e., 30 min of continuous physically active at least 3 times per week) males were recruited to participate in this study. Potential subjects were excluding if they were taking any dietary supplements, using any prescribed or recommended medications, reported known history of any disease or condition requiring medical attention, suffered a lower leg injury during the previous year that required the use of crutches, planning on increasing or decreasing the amount of time spent in the sun or tanning bed, or traveling south of 37° N in latitude during study participation. Subjects were also excluded if they were morbidly obese (body mass index (BMI) > 40 kg/m2) or displayed strength or power output asymmetry (i.e., > 5% difference in peak isometric or power output between legs). Symmetry in leg strength was important since all strength testing and the exercise protocol (see below) were performed unilaterally and statistical comparisons were made within and between legs. Data was collected during the winter (between November and March) in Salt Lake City, UT, USA (40° N latitude).

Study design and protocol

This study consisted of a randomized, double-blind, placebo-controlled experimental design. Subjects were randomly assigned to one of two groups: (1) placebo (n = 13) or (2) vitamin D (cholecalciferol, 4000 IU; n = 15) supplementation. Supplements (donated from USANA Health Sciences, Inc.) were taken daily for 35-d. During participation, subjects were asked to keep their diet consistent with their regular eating habits during the previous year and to refrain from using any dietary supplements. Subjects were also asked to refrain from physical activity, and using aspirin, ibuprofen, naproxen sodium, acetaminophen, or other anti-inflammatory agents 72-h prior to a blood draw.

Each subject provided eight fasting blood samples: (1) baseline, (Bsl and 28-d before the exercise protocol), (2) immediately before [Pre] and (3) immediately [Post], (4) 1-h, (5) 24-h, (6) 48-h, (7) 72-h, and (8) 168-h after the exercise protocol. Single-leg strength testing and perceived muscle soreness were performed on seven different occasions: (1) Bsl, (2) Pre, (3) Post, (4) 24-h, (5) 48-h, (6) 72-h, and (7) 168-h. Each visit commenced within 1-h of waking and between 06:00 and 09:00 a.m. The time remained consistent within each subject across all visits. Perceived muscle soreness was performed before the blood draw procedure, and the blood draw was performed prior to strength testing. Supplementation started at Bsl after the blood draw, perceived muscle soreness, and single-leg strength testing procedures.

Exercise induced muscle damage

Each subject had one randomly selected leg (stretch-shortening contraction; SSC) perform an intense exercise protocol. The other leg served as the contralateral control (CON). The exercise protocol was performed 28-d after Bsl and consisted of 10 sets of 10 repetitive eccentric-concentric jumps at 75% of body mass with a 20 sec rest between each set, as described elsewhere[14, 17]. Briefly, subjects were instructed and verbally encouraged to perform each set with maximal effort and to jump as-high-as possible through a full range of motion (90° of knee flexion-to-full extension). If subjects were no longer able to complete two-successive jumps, subjects were then allowed to perform presses (foot remaining on the force plate) through a full range of motion (90° of knee flexion-to-full extension). If subjects were unable to complete two successive presses, the exercise protocol was terminated. The rationale for including the presses was to allow the subjects to successfully complete the requested number of sets and repetitions. However, switching from jumps to presses presumably lowered the eccentric magnitude during loading and some subjects were unable to complete the protocol.

The mean number of jumps and presses completed during the exercise protocol were not significantly (statistically) different between the placebo (jumps, 62 ± 9; presses 19 ± 4) and vitamin D (jumps, 59 ± 8; presses, 30 ± 6) groups. Six (46%) of the placebo subjects and 9 (60%) of the vitamin D subjects completed the exercise protocol. Blood chemistries, perceived muscle soreness, and leg strength data were not significantly different between those who finished and those who did not finish the exercise protocol or between the placebo and vitamin D groups as determined by a repeated measures analysis of variance (ANOVA).

Blood sample handling

Fasting blood samples were obtained from the antecubital vein into one 4.0 mL purple-top Becton Dickinson (BD; Franklin Lakes, NJ, USA) Vacutainer tube (K2 EDTA 7.2 mg plasma), one 4.5 mL green-top BD Vacutainer tube (PST Gel and lithium heparin, 83 units), and one 6.0 mL red-top serum BD Vacutainer tube. Plasma was separated by centrifugation (Heraeus Labofuge 400 series, Buckinghamshire, England) at 2400 g for 6 min within 20 min of sample collection. Following separation, plasma samples were sent to ARUP Laboratories (Salt Lake City, UT USA) for clinical chemistries (see below). After coagulation, serum was separated by centrifugation (VWR International, Clinical 50 Centrifuge) at 1100 g for 20 min, and then aliquoted into several small micro-centrifuge tubes. Aliquoted serum samples were stored at -80°C (Revco Freezer, GC Laboratory Equipment, Asheville, NC, USA) until analysis (see below).

Analytical procedures

Serum 25(OH)D concentrations

Serum 25(OH)D concentrations (ng/mL) were measured in duplicate (coefficient of variation (CV) = 3.90%) in each blood sample, as described previously[18]. Briefly, analytes were separated on an Agilent high performance-liquid chromatography system (series 6460, Model G6460A, Santa Clara, CA, USA) and detected on an Agilent tandem mass spectrometer (Series 6410, Model G6410B, Santa Clara, CA, USA) using atmospheric pressure chemical ionization (APCI) detection (350°C gas temperature, 400°C vaporizer). The 25(OH)D 3 , deuterated 25(OH)D 3 internal standard, and 25(OH)D 2 precursor ions were 383.3, 386.3, and 395.4, respectively. The 25(OH)D 3 , deuterated 25(OH)D 3 , and 25(OH)D 2 product ions were 365.3, 368.3, and 208.9, respectively. Serum 25(OH)D 2 and 25(OH)D 3 concentrations were determined relative to authentic standards and corrected for recovery of the 25(OH)D 3 internal standard. Serum 25(OH)D 2 (limit of detection = 2.0 ng/mL) was not detected in any of the subjects, and therefore, serum 25(OH)D total concentrations are referred to as serum 25(OH)D concentrations hereafter.

Clinical chemistries

A comprehensive metabolic panel was performed on Bsl plasma samples. Plasma AST (U/L), ALT (U/L), PTH (pg/mL), and calcium (mg/dL) concentrations were measured in each blood sample (ARUP Laboratories, Salt Lake City, UT, USA).

Single-leg strength testing

Single-leg strength testing was performed on a horizontal Plyo-Press (Athletic Republic, Park City, UT, USA), as described previously[17–19]. In brief, the Plyo-Press sled was adjusted for each subject to align the knee and hip joint flexion angles to 90° with the abdominal, low back region secured and stabilized to the Plyo-Press sled with a harness. Subjects performed submaximal isometric contractions on each leg at increasing intensities (≈ 50, 75, and 90%) prior to performing the testing contractions to become familiar with the testing protocol and procedure. Leg selection (i.e., CON or SSC leg) at the start of each testing session was randomized and followed by an alternating sequence of leg contractions. Peak isometric contractions (i.e., hip and knee extension) on each leg were performed in triplicate (CON leg CV = 2.62%; SSC leg CV = 6.13%). Each isometric contraction was 3 sec in duration and separated by 1 min of rest. Subjects were verbally instructed and strongly encouraged to exert maximal force against the mounted force platform. Peak isometric force was defined as the highest resultant force produced from the three isometric tests and expressed relative to body mass (N/kg).

Single-leg peak power output measures followed the single-leg isometric contractions and were measured on the same horizontal Plyo-Press with the same securing procedures described above. Starting from an extended position (full extension = 0°), subjects performed several submaximal jumps to become familiar with the testing protocol and procedure. Thereafter, subjects performed repetitive maximal effort single-leg jumps (i.e., hip and knee flexion-extension cycles). Subjects were instructed and verbally encouraged to perform the jumps as fast as possible and through a full range of motion (90° of knee flexion-to-full extension). Each test was 20 sec in duration with the weight-stack resistance set at 75% of body mass. The time-aligned product of the resultant force (N) acquired from the force platform and the weight-stack velocity (m/s) data obtained from the displacement transducer were used to calculate power output. Peak power output was defined as the highest power output produced during the 20 sec test for each leg.

Plyo-Press output data were measured from signals obtained from a mounted force plate (Advanced Mechanical Technology, Watertown, MA, USA) and from a displacement transducer (UniMeasure PA-50-NJC, Corvallis, OR, USA) attached to the weight stack. Data were sampled at 200 Hz with a low-pass filter at 10 Hz using DartPower software (Athletic Republic, Park City, UT, USA, version 2.0).

Muscle soreness

To assess muscular soreness, subjects lowered their body into a squat position (90° of hip and knee flexion) for 5 seconds while resting their back against a wall. This was performed in triplicate at each visit. In the squat position, subjects rated the perceived soreness of their gluteus, quadriceps, hamstrings, and calves in each leg by using a visual analog scale (10 cm in length) of 1 to 10, with 0 being ‘no pain’, 10 being the ‘worst possible pain’, and 5 being ‘tender to touch but not to contractions’[20]. Subjects made a perpendicular mark on the visual analog scale from which perceived soreness was quantified by measuring to the nearest tenth of a centimeter.

Statistical analyses

Data were checked for normality prior to all statistical analyses with a Shapiro-Wilk test. Statistical significance of subject characteristic and exercise protocol data (i.e., number of jump and press repetitions) were assessed using a two-sample t-test with a Bonferroni adjustment. Statistical significance of data (i.e., serum 25(OH)D, calcium, PTH, peak isometric force, and peak power output) were assessed using a repeated-measures ANOVA and post hoc t-tests with a Bonferroni correction on multiple pairwise comparisons when appropriate. Due to non-normally distributed data, statistical significance of ALT, AST, peak force, and perceived muscle soreness (gluteus, quadriceps, hamstrings, and calves) data were assessed with separate Friedman ANOVA tests. Statistical significance in strength recovery were assessed with a two-sample t-test (peak power output) with a Bonferroni correction for multiple pairwise comparisons or with a Wilcoxon Signed-Ranked Test (peak isometric force) if data were non-normally distributed. Relationships between variables were assessed with a Pearson product-moment linear correlation. All statistical analyses were performed with SYSTAT (version 13.1, Chicago, IL, USA). Statistical significance was set at P < 0.05. Data presented as mean (SD) unless noted otherwise.