Subjects

The institutional review board approved the study and participants provided written informed consent prior to testing and participation. A total of twenty-four participants from the local area completed this study (Table 1). To be eligible participants had to be: male; aged 18–36; have no acute or chronic medical conditions; have the ability to bench press 100% bodyweight; and had been performing regular resistance training exercise for at least three days per week for the previous two years. None of the participants were supplementing their diet with any ergogenic or testosterone booting supplements prior to testing. All participants provided written consent and completed a medical history check. The study was approved by the University of Western Sydney human research ethics committee, and carried out in accordance with the declaration of Helsinki.

Table 1 Participant demographics Full size table

Experimental approach to the problem

This was a randomised, double-blinded, and placebo-controlled design to examine the effects of d-aspartic acid supplementation on basal testosterone levels following a two week supplementation protocol. Participants were assigned to one of three experimental groups: placebo (D0), three grams of DAA (D3) and six grams of DAA (D6). All participants consumed 10 opaque capsules each morning with breakfast for two weeks. They contained either: six grams of flour (D0, n = 8); a mixture of three grams each of flour and DAA (D3, n = 8); or six grams of DAA (D6, n = 8). Participants were randomly allocated to treatment groups following a block randomisation procedure based on a computer-generated list of random numbers. Placebo, mixed and supplement were provided in identical opaque capsules to improve blinding. Group allocation was managed by a technical officer, whilst investigators were kept blind to group assignment throughout the intervention. All participants followed an upper/lower body split resistance training program for a full month, with the initial two weeks of training (washout period) performed without supplementation (Figure 1). Three timepoints were used to obtain testing data: T1, T2 and T3 (Figure 1).

Figure 1 Timeline of the study. After completion of T1, subjects began training four days per week. Daily supplementation commenced after T2 ( ). T1-3 included fasted blood draws ( ). Full size image

Experimental procedures

Testing sessions consisted of a fasted blood draw, then 1-RM bench press evaluation. Initial baseline blood measures were taken at two timepoints (T1 & T2) and averaged to ensure accuracy in baseline assessment of these markers (Figure 1). After T1 prescribed training commenced for four weeks. After testing session T2 daily supplementation begun with training continuing as before. Post-measures were taken after these last two weeks of training and supplementation, at the end of week 4 (Figure 1). The supplemental period of two weeks was chosen as this has been previously shown to be a sufficient time period to see a change in total testosterone levels [12].

1-RM testing

Bench press dynamic strength one repetition max (1-RM) was measured before the standardisation period (T1), beginning of experimental period (T2) and post experiment period (T3) (Figure 1), as part of eligibility testing. Correct form included depth to the level of the chest, with feet not leaving the floor, and the backside not leaving the bench at any point during the repetition. The protocol for 1-RM testing involved one warm up set of 10 reps at approximately 50% of their estimated 1-RM, followed by two more warm ups at approximately 70% and 80% with only 1–2 reps. After the warm ups participants attempted 1-RMs with incrementally increasing weight. The weight achieved prior to the failed attempt was recorded as the 1-RM. A participant’s 1-RM was achieved within five attempts and adequate rest between attempts was adhered to (3–5 mins) [18].

Fasted blood draws

All blood draws were obtained via venepuncture of the antecubital vein after a 12 hour fast. Participants were also instructed to avoid strenuous exercise and alcohol consumption the day before the draw. Blood draws were conducted by a trained phlebotomist and subsequent draws were planned for the same time of morning (7:00–10:00 am) for each particular participant, to prevent any effect of diurnal variation. Whole blood was collected using serum separator tubes (SST™ II Advance, BD Vacutainer®). They were then allowed to clot for 45 minutes and centrifuged using a fixed angle rotor centrifuge: ADAMS® Compact II Centrifuge, V:227 (Becton Dickinson & Co) (828 × g, at 2700 rpm) for 15 minutes in an air conditioned room (19°C). Serum was aliquoted and stored at −80°C until analysis (Douglas Hanly Moir Pathology, Macquarie Park, NSW, Australia). Single analysis of serum was conducted for total testosterone, estradiol, sex-hormone-binding-globulin (SHBG) and albumin. Testosterone and SHBG was measured via electrochemiluminescent (ECL) immunoassay, on a Roche E170 system (Roche Diagnostics). Albumin was measured via bromocresol green (BCG) succinate buffer method, on an Abbott 16000. Estradiol was measured via chemiluminescent microparticle immunoassay on an Abbott i2000. Free testosterone was calculated from total testosterone, SHBG and albumin.

Training standardisation

Participants trained for four days per week over a one month period. The prescribed training for each exercise consisted of four sets of a repetition maximum range of 8–10. If the repetition range wasn’t met, participants were asked to lower or raise the weight in the next session. Exercises during the upper body session were: barbell bench press; overhand pulldown; barbell overhead press and underhand pulldown. The lower body session consisted of: back squat; good morning; leg extensions; and straight leg calf raises. Adherence was monitored via training diaries and supervised sessions (minimum 1 × per week).

Dietary intake

Participants were asked to control their diet, by avoiding any major changes throughout the study duration. To monitor their diet they were asked to weigh and recorded their food intake for three days each of the first and last week; two training days and one non-training day. These three days were averaged to get a daily mean for week one and four. The food diaries were entered into CalorieKing (Australian Edition 4.0), then analysed for caloric and macronutrient daily intakes (protein, carbohydrates and fats) and normalised to bodyweight.

Statistical analysis