The results of this paper indicate that DAA supplementation is ineffective at changing testosterone levels, or positively affecting training outcomes. Reductions in estradiol and the blunting of peripheral excitability appear unrelated to improvements from resistance training.

No change in basal TT or FT were observed after the intervention. DAA supplementation (n = 10) led to a 16%, 95% CI [-27%, -5%] reduction in E 2 from T1-T3 (p<0.01). The placebo group (n = 9) demonstrated improvements in spinal responsiveness (gastrocnemius) at the level of the alpha motoneuron. Both groups exhibited increases in isometric strength of the plantar flexors by 17%, 95% CI [7%, 28%] (p<0.05) as well as similar increases in hypertrophy in the quadriceps and calf muscles.

Research on d-aspartic acid (DAA) has demonstrated increases in total testosterone levels in untrained men, however research in resistance-trained men demonstrated no changes, and reductions in testosterone levels. The long-term consequences of DAA in a resistance trained population are currently unknown.

Data Availability: The study was approved by the Western Sydney University Human Research Ethics Committee and data are ethically restricted to statistical reporting for reasons relating to patient privacy. Relevant data can be found in the Figshare Repository at the following URL: https://figshare.com/articles/The_effects_of_d-aspartic_acid_supplementation_in_resistance-trained_men_over_a_three_month_training_period/5280592 . The DOI is 10.6084/m9.figshare.5280592 .

Copyright: © 2017 Melville et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The primary objective of this study was to evaluate the effectiveness of DAA to alter basal testosterone levels over three months of resistance training. A secondary objective was to establish potential mechanisms for changes in strength and hypertrophy. Based on our previous findings, it was hypothesised that the DAA group would experience decreased total testosterone and free testosterone. In addition, it was hypothesised that the DAA group would experience decreased strength, which would be explained by changes in the corticospinal pathway.

The potential adverse effects of DAA on testosterone, in addition to affecting hypertrophic outcomes, may also have an effect on the neural mechanisms associated with increased muscular strength. Strength appears to be a function of both the size [ 13 ] and composition of muscle [ 14 ], in addition to the rate and magnitude of output from the nervous system [ 15 , 16 ]. Resistance training increases the voluntary activation of the nervous system [ 17 ], in addition to increasing the input-output response of cells in the spinal cord [ 18 – 20 ]. There is a growing body of research that suggests DAA fills the criteria of a neurotransmitter [ 21 – 23 ], which potentially could provide strength improvements via increased neurotransmitter availability. Neural plasticity, which is observed in untrained individuals is a key explanatory mechanism of strength development [ 24 ], and can involve cells at the spinal and supraspinal level. Measures of neural plasticity that examine spinal and supraspinal output, such as the H-reflex and V-wave respectively, have not been measured following a period of resistance exercise in trained individuals and as such, it is not clear if trained populations have the same neural adaptation response to resistance training as untrained individuals. The link between testosterone and changes in the corticospinal pathway has been explored in humans with artificially induced testosterone levels [ 25 ]. These increases in testosterone were concurrent with reduced threshold of the evoked potentials from transcranial magnetic stimulation (TMS), indicating that testosterone can positively increase the output from a given input, within the corticospinal pathway [ 25 ]. It is plausible that artificially changing testosterone levels, via supplementation may affect strength or power, by altering the efficiency of the corticospinal pathway. The effects of DAA on neural plasticity has yet to be researched in humans.

Early human research demonstrated that three grams per day of DAA supplementation increased total testosterone in untrained men by ~42% [ 6 , 7 ]. Subsequently the popularity of DAA supplementation in recreational resistance training has increased, owing to the positive relationship between testosterone levels and mechanisms of muscular hypertrophy, such as increased protein synthesis [ 8 , 9 ] and satellite cell proliferation [ 10 ]. However in resistance-trained men, three grams of DAA per day resulted in no meaningful change in testosterone levels, or training outcomes after 14 [ 11 ] or 30 days of supplementation [ 12 ]. Indeed, Melville and colleagues reported that a larger daily dose of DAA (6 g.d -1 ) actually decreased basal testosterone by ~12.5% after 14 days of supplementation, suggesting a deleterious effect on negative feedback mechanisms of the HPG axis [ 11 ]. This reduction is a particularly concerning finding considering the supposed importance of basal testosterone, with respect to hypertrophic training outcomes. It is unclear if the observed decline in basal testosterone after 14 days will continue if supplementation is continued for a longer timeframe (i.e. three months). Moreover, the relationship to training outcomes if testosterone continues to decline, or otherwise is maintained at a new basal level, is unknown.

D-aspartic acid (DAA) is an amino acid that exists in central nervous and reproductive tissues. Animal and human research suggest that DAA functions in the development of the nervous system as well as hormonal regulation [ 1 , 2 ]. Data in mammalian studies indicate that DAA supplementation can influence the hypothalamic-pituitary-gonadal axis (HPG) at the level of the hypothalamus [ 3 ], anterior pituitary [ 3 , 4 ] and the testes [ 4 , 5 ]. Accumulation of DAA at these sites is associated with an upregulation of testosterone production in these animals, as well as upstream effectors of the HPG axis (i.e. luteinizing hormone).

Methods

Subjects The present study was approved by the Western Sydney University Research Ethics Committee (H10087) and data are ethically restricted for reasons relating to participant privacy. All relevant data are contained within the paper and supporting files. All participants gave written consent and completed a medical history check. The study was carried out by the Declaration of Helsinki. Participants were recruited from the University campus via flyers, lecture announcements and online intranet advertising. In addition, recruitment from the western Sydney area was promoted via Facebook advertising. In response to recruitment 31 people were assessed for eligibility. Twenty-two subjects were recruited, three dropped out due to personal reasons, leaving 19 that completed the study (Fig 1). A rolling recruitment strategy was conducted from 17th of March to the 16th of December 2014, with participants beginning soon after they were recruited, thus participants began the study at different time periods. Final testing was conducted at the end of the 12 week training period and the trial was ended according to a set schedule for each participant. The last day of data collection was the 10th March 2015. The sample size for the present study was determined from the reduction in free testosterone observed in previous DAA research [11]. Free testosterone changes in the six-gram group demonstrated an effect size of 1.17, which required 12 participants to be adequately powered. A Means statistical test (matched pairs) using G Power v 3.1. was used to estimate effect size using group parameters from the previous research [11]. Power calculations were determined A Priori with an alpha level of 0.05 and two tailed significance. Participant demographics are presented in Table 1. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Comparison of participant characteristics for 6 g/d of d-aspartic acid (DAA) and placebo (PLA), presented as mean±SD. https://doi.org/10.1371/journal.pone.0182630.t001 PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Consort flow diagram. https://doi.org/10.1371/journal.pone.0182630.g001 Inclusion Criteria: aged 18–36; have no acute or chronic medical conditions; have the ability to bench press 100% bodyweight; had been performing regular resistance training exercise for at least three days per week for the previous two years; and not supplementing diet with any ergogenic or testosterone boosting supplements.

Study design The present study was a randomised, double-blinded, and placebo-controlled research design. Subjects attended three laboratory sessions, located at the University campus, baseline testing (T1), midpoint testing (T2) and post testing (T3). These testing sessions involved: fasted blood draws for serum hormonal analysis; Brightness-mode (B-mode) ultrasound of the quadriceps and calf muscles for hypertrophy changes; electrical stimulation of the tibial nerve to determine H-reflex and V-wave responses of the plantar flexors; and isometric maximal voluntary contraction (MVC) of the plantar flexor muscles. Following T1, all participants followed a structured resistance training protocol, which was combined with their daily supplementation protocol. Participants were assigned to one of two experimental groups: placebo (PLA, 6g rice flour, n = 11) or d-aspartic acid (6g DAA, n = 11). Three participants were lost to follow-up, thus analysed participants included PLA, n = 9; DAA, n = 10. One participant from the placebo group did not have sufficient data for plantar flexor strength and neural measures, thus was omitted from those analyses. All participants consumed 10 identical opaque capsules each morning with water before they had breakfast for 12 weeks. Adherence to daily supplementation was controlled for via prompts at supervised training sessions, in addition the number of pills left were compared to their set schedule each week. Participants were randomly allocated to treatment groups following a block randomisation procedure (block size of four) based on a computer-generated list of random numbers. Group allocation was managed by a technical officer, while the primary investigator and participants was kept blind to group assignment throughout the experimental intervention, and data analysis. The protocol as explained in the original participant information sheet, included 1RM testing for the squat and bench press. These were omitted due to time constraints of participants on testing days, as well as the fact that isometric testing would provide a stronger match with neural testing of the plantar flexors. As this study utilised a commercially available supplement rather than a drug, it was not expected that this study would fit the definition of a clinical trial, therefore the clinical registration of this study was performed retrospectively (ANZCTR).

Training protocol Participants trained four days per week, with supervised sessions conducted a minimum of once per week. The participants training was monitored using training diaries. The prescribed training involved five exercises that included 3–5 sets of various repetition maximum (RM) prescriptions (2RM– 10RM). A primary lift was prescribed for each day–deadlift, bench press, good morning/stiff-leg deadlift and squat, with accessory exercises for balance and volume. These accessory exercises included: seated rows, pull-down, bent over row, biceps curl, chin-ups, bench press, overhead press, pec fly, dip and back extension. See Table 2 for programming of measurement specific exercises. PPT PowerPoint slide

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larger image TIFF original image Download: Table 2. Program design for exercises that primarily targeted the quadriceps and calf muscles. Prescribed training was either repetition maximum (RM) or body weight to failure (BWF). https://doi.org/10.1371/journal.pone.0182630.t002 Periodisation changes were implemented at week six to improve motivation and prevent overtraining. These changes can be observed in Table 2, with an increase in training intensity with some of the exercises, but they did not affect the overall layout of the protocol. All participants were asked to perform 48 sessions in the gym in total (average number of sessions performed 47.5±0.8, an adherence rate of 99.0%).

Testing sessions During laboratory sessions, the subject was asked to lie down upon arrival. After checking for pre-testing adherence protocols (fasting status) participants were asked to lie down for two minutes. Blood was then extracted from the antecubital vein via venepuncture. Participants remained lying down for 20 minutes before ultrasound procedures followed. Following ultrasound testing, the subject was seated into an isokinetic dynamometer (KinCom 125, version 5.32, Chattanooga, TN, USA). After the KinCom was configured, the subject was prepared for EMG and the cathode location was determined for transcutaneous nerve stimulation. The protocol for the determination of isometric plantar flexor strength, H-wave, M max and V-waves followed. The overview of the study protocol is visually represented in Fig 2. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. Testing procedures overview. In chronological order, the study protocol comprised of: Consent and forms; venepuncture blood draws; ultrasound testing of the quadriceps and calf muscles; EMG and stimulation preparation; isometric MVCs (plantar flexors); and determination of H-wave curve and V-waves via electrical stimulation. https://doi.org/10.1371/journal.pone.0182630.g002

Fasted blood draws Participants were instructed to fast for 12-hours and avoid strenuous exercise and alcohol consumption the day before testing. For each participant, the initial blood draw was scheduled in the morning between 7:00–10:00, with subsequent draws time-matched on an individual basis. Whole blood was collected using serum separator tubes (SST™ II Advance, BD Vacutainer®), and rested for 45 minutes in an air-conditioned room (19°C). Collection tubes were centrifuged using a fixed angle rotor centrifuge (ADAMS® Compact II Centrifuge, V:227, Becton Dickinson & Co), run for 15 minutes at 2700 rpm and 828 x g. Serum was aliquoted and stored at ‒80°C until analysis by an external laboratory (Douglas Hanly Moir Pathology, Macquarie Park, NSW, Australia). A single analysis of serum was performed for total testosterone (TT), estradiol (E 2 ), sex-hormone-binding-globulin (SHBG) and albumin (ALB). Testosterone and SHBG were measured via electrochemiluminescence immunoassay, on a Roche E170 system (Roche Diagnostics, Dee Why, NSW, Australia), with a limit of detection values of 0.025 ng/ml and 0.350 nmol/l respectively. Albumin was measured via bromocresol green succinate buffer method (BCG), on an Abbott ARCHITECT c16000 (Abbott Laboratories, Abbott Park, Illinois, USA). The limit of detection of the ALB assay is 30 g/l. Estradiol was measured via chemiluminescent microparticle immunoassay, on an Abbott i2000 (Abbott Laboratories, Abbott Park, Illinois, USA) and the analytical sensitivity of this assay is ≤ 10 pg/ml. Free testosterone (FT) was calculated from TT, SHBG and ALB [26].

Ultrasound Participants were asked to rest in a supine position for 20 minutes, to allow for the fluid shift in the muscles to stabilise [27]. Extended-field-of-view (EFOV) images were conducted at the inferior third of the quadriceps muscles, measured as a third of the distance between the centre of the knee joint and the Anterior Superior Iliac Spine (ASIS). B-mode ultrasound imaging was conducted using an Echo Blaster 128 family scanner and Echo Wave II v2.3.6 (Telemed, Vilnius, Lithuania) software. Quality control methodology has been previously outlined [28]. These images were analysed for Cross Sectional Area (CSA) of the quadriceps muscles; vastus lateralis (VL), vastus intermedialis (VI), vastus medialis (VM) and rectus femoris (RF). A minimum of three images was obtained for each time point, with the average values from these images used for data analysis. Images were analysed using ImageJ 1.46 (National Institutes of Health, Bethesda, Maryland, USA) public domain software package. Intra-experimenter reliability (CV) of the EFOV method was 2.26%. Muscle thickness was determined for VL and VI at the inferior third (33%) and the midpoint of the quadriceps (50%). To obtain sagittal images the probe was manipulated until the superficial, and deep aponeuroses were parallel, and the pennation fibres were straight rather than curved. Muscle thickness was obtained for vastus lateralis (VL 33%, VL 50% ) and vastus intermedialis (VI 33%, VI 50% ). Pennation angle for vastus lateralis (VL Angle ) was determined at the midpoint quadriceps site (50% between the centre of the knee joint and ASIS). To determine calf muscle thickness, the distance between the lateral malleolus and the fibular head was divided into three sites: Superior quarter, SOL 75% and GAS 75% ; superior third, SOL 67% and GAS 67% ; and midpoint, SOL 50% and GAS 50% . Intra-experimenter reliability (CV) of the analysis of the sagittal images was 2.99% for the quadriceps thickness, 8.68% for calf thickness and 13.67% for pennation angle.

EMG Electromyography was recorded from the soleus and gastrocnemius muscles using bipolar Ag/AgCl electrodes (Maxensor; Medimax Global, Australia). The skin was prepared following standardised procedure to reduce impedance below five kΩ. Soleus electrodes were placed at two-thirds of the distance between the medial condyle of the femur and the medial malleolus. Two electrodes were placed on the muscle belly for the medial gastrocnemius site, and the medial malleolus landmark was used for the reference electrode. Placement of the electrodes was recorded to ensure consistency between testing sessions. A ML138 Octal BioAmp (common mode rejection ratio >85 dB @ 50 Hz, input impedance 200 MΩ) with 16-bit analog-to-digital conversion, sampled at 2 kHz (ADI Instruments, Sydney, NSW, Australia) was used to record sEMG signals into LabChart v7.3.7. Raw signals were filtered with a fourth-order Bessel filter between 20–500 Hz.

Isometric force Isometric testing was chosen over dynamic 1RM strength testing, as it was believed to be a stronger and more reliable match to the neural protocol. The isokinetic dynamometer (KinCom 125, version 5.32, Chattanooga, TN, USA) was configured, so the seat was set upright, with the participant’s hip and knee flexed to 90° degrees and the subject’s lateral malleolus in line with the centre of rotation of the lever arm. The seat angle was adjusted so that there was no gap between the knee and the edge of the seat to prevent unwanted muscle activity. The participants left foot was attached to the plantar-flexion–dorsiflexion attachment with hook-and-loop straps. Two seatbelts were applied across the thighs and chest. Subjects were warmed up with three submaximal contractions, ~50%, ~75%, ~90% Maximal Voluntary Contraction (MVC) spaced one minute apart. After sufficient warm up, three MVCs were recorded, with two minutes rest given between each MVC. Subjects were instructed, from complete relaxation, to contract their plantar flexors as hard and as fast as possible, holding the contraction for five seconds. Verbal encouragement was given during MVC attempts. This was standardised by repeating the word “GO” during the five seconds of contraction. Visual feedback of the instantaneous torque production was displayed on a screen that participants could see. Torque output signals were directly sampled from the dynamometer at 2 kHz (PowerLab, ADInstruments, Sydney, NSW, Australia). MVC was defined as the peak isometric torque (N.m) exerted within the 5s force trace.

Posterior tibial nerve stimulation H-reflexes, M-waves and V-waves were evoked from the soleus and gastrocnemius using a 1-ms square wave pulse, delivered by a constant current stimulator (DS7AH Stimulator, Welwyn Garden City, UK) at 400 V, applied to the tibial nerve. A custom anode (Aluminium foil 10x6 cm and electrode paste) was applied to the anterior aspect of the thigh, proximal to the patella. A single Ag/AgCl electrode (Maxensor; Medimax Global, Australia), was located on the posterior side of the left knee within the popliteal fossa. To determine nerve location a rubber-insulated probe was manipulated within the popliteal fossa until the largest evoked resting H-wave was observed from the soleus EMG trace. To map the H-M curve, H-wave threshold was approximated by increasing stimulation intensity from 10 to 30 mA (1 mA increments). The start of the H-M curve was defined as three mA lower than the intensity to evoke ≥ 1 mV. To approximate M max , the stimulus intensity was increased by 10 mA increments until a plateau was achieved over three stimulation points, with the last point defined as M max . Thirty stimulation points were created on a logarithmic scale between these two approximations. Two stimulations (with 10 seconds rest between) were recorded for each of the thirty points.

H-wave data processing For H-wave processing, the ascending limb was defined as data from the initial stimulation point to H max . Data from the ascending limb was entered into a custom program coded in the statistical package R (R Foundation for Statistical Computing, Vienna, Austria). This program incorporated a three-parameter sigmoid function and used a general least squares model to find the best fit for the ascending limb H-wave data (Klimstra & Zehr, 2008). Recruitment curves with an R2 < 0.90 were omitted from analysis (Vila-Cha et al., 2012). The following parameters were acquired from the ascending limb: the slope at the midpoint (H slp ), current at H-wave threshold (iH th ), current at H-wave maximum amplitude (iH max ) and current at 50% H max (iH 50 ). These parameters are visually represented in Fig 3. V-wave data was processed by normalising the value to the maximal motor response for that time point (M max ). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. H-reflex parameters displayed on a representative example. The raw data is represented as open circles, with the sigmoid fit data represented along the solid line. The short dashed line represents the slope at 50% of H max . The lower filled circle represents iH th , and the upper filled circle represents iH max . https://doi.org/10.1371/journal.pone.0182630.g003