Vitamin D is a micronutrient that also acts as a prohormone ^[1][112]. Vitamin D has garnered considerable attention in the general and competitive athletic populations, primarily due to its role on various physiological systems in the body, and the effect that vitamin D deficiency has on many diseases ^[2][3][113,114]. Vitamin D has two biological forms, vitamin D3 (cholecalciferol), and vitamin D2 (ergocalciferol). Vitamin D3 is the most bioavailable and most supplemented form of Vitamin D and is synthesized in the skin upon exposure to sunlight. The specific physiological effects of vitamin D and its specific mechanisms are beyond the scope of this paper, but readers are encouraged to explore this elsewhere ^[4][5][115,116]. In brief, vitamin D, whether it is synthesized endogenously or consumed as a food or supplement, undergoes hydroxylation to become active. The first hydroxylation step occurs in the liver where vitamin D is converted to 25-hydroxyvitamin D [25(OH)D]. The second hydroxylation step is performed primarily in the kidney to form 1,25-dihydroxyvitamin D3, also referred to as 1,25-dihydroxycholecalciferol, which is the biologically active form of vitamin D ^[6][7][117,118]. The United States Institute of Medicine has indicated that the range for vitamin D concentrations should be between 25–50 nmol·L^{−1 [8]}[119]. Studies on athletes have suggested that the cut-off for vitamin D deficiency should be > 30 nmol·L⁻¹ or even higher ^[1][9][10][112,120,121].

The vitamin D receptor, which plays a central role in the biological action of the vitamin, has been observed in reproductive tissues such as the ovaries, prostate, and testes, as well as in human sperm ^{[11][12][13][14]}[122,123,124,125]. Vitamin D receptors are present on the Leydig cells within the testes, where the synthesis of testosterone from cholesterol occurs ^[15][7], suggesting an important role of vitamin D on testosterone synthesis. Men with vitamin D deficiency have exhibited significantly lower testosterone concentrations compared to men with normal vitamin D concentrations ^[16][126]. Significant associations were also noted between vitamin D concentrations and circulating testosterone and SHBG concentrations, as well as the free androgen index ^[16][126]. These findings are consistent with subsequent investigations reporting significant correlations between vitamin D and testosterone concentrations ^[16][17][18][126,127,128].

Athletes in general are at a higher risk for vitamin D deficiency, especially athletes participating in indoor sports ^{[10][19][20][21]}[121,129,130,131]. Vitamin D supplementation is a potential option to maintain normal vitamin D status, but also to potentially increase testosterone concentrations. A double-blind, randomized placebo-control trial of 54 males reported that the group receiving a daily supplementation of 83 μg (3332 IU) of vitamin D for 12 months experienced significant increases in circulating 25-hydroxyvitamin D, TT, and FT concentrations compared to the placebo group ^[22][132] (described in Table 1). Although it has been suggested that the daily dose of vitamin D supplementation for athletes should be 5000 IU·day⁻¹ for improving performance and restoring vitamin D levels ^[9][23][24][120,133,134], no consensus exists regarding the optimal range for vitamin D levels ^[8][119]. Furthermore, the effect of vitamin D supplementation on altering resting testosterone concentrations is still not well understood and requires further research.

Zinc is a mineral that influences and interacts with many biological systems, especially the endocrine system ^[25][16]. Zinc has an important role in immune system function and in modulating inflammatory processes ^{[26][27][28][29]}[135,136,137,138]. While zinc can be found in many food sources, the more bioavailable form of zinc can be found in animal tissues ^{[30][31][32][33]}[139,140,141,142]. The daily recommended intake for zinc is between 14–40 mg·day^{−1 [34]}[143]. The physiological role of zinc regarding testosterone biology is related to its requirement in the synthesis and secretion of LH. As previously discussed, LH stimulates testosterone synthesis in the Leydig cells ^[35][36][37][6,14,144]. Zinc is also important in the conversion of testosterone to DHT ^[37][144]. DHT is converted from testosterone by the enzyme 5α—reductase in the cytoplasm of the cell. DHT is primarily found in peripheral tissues such as prostate, skin, hair follicles, and the liver ^[35][38][6,145]. As discussed earlier, DHT is thought to have a stronger androgenic affect than testosterone due to its four-times greater binding affinity for the androgen receptor than testosterone and a three-times slower dissociation rate than testosterone ^[39][40][41][146,147,148]. DHT has a vital role in the sexual development of males and sexual differentiation of organs and promotes prostate growth; male pattern baldness; and body, facial, and pubic hair growth ^[35][38][42][6,145,149].

Zinc also has an indirect role in testosterone synthesis. Zinc is required for normal function of angiotensin-converting enzyme (ACE), a zinc-dependent dicarboxypeptidase, which has a zinc binding site in its cyclitic domain ^[43][44][150,151]. ACE is reported to increase LH production in pituitary, thus impacting androgen production ^[45][152]. Zinc deficiency can impair testosterone synthesis and has been demonstrated to correlate with reductions in testosterone concentrations ^[46][47][48][153,154,155]. Competitive athletes appear to be at a greater risk for zinc deficiency compared to the general population ^[49][50][156,157]. Considering that zinc deficiency appears to be related to hypogonadism, efforts to maintain zinc levels within normal ranges appears important. Several studies have shown that zinc supplementation can restore testosterone concentrations to their normal physiological range ^[46][47][51][153,154,158]. One study examined the effect of zinc supplementation on both TT and FT concentrations in healthy young adults before and after an exhaustive exercise protocol ^[52][159]. Study participants were supplemented with zinc sulfate (3mg·kg·day⁻¹) for four weeks. Investigators reported that zinc supplementation increased both TT and FT concentrations prior to and following the exhaustive exercise protocol compared to pre-supplementation results. In contrast, others reported no difference in either the TT or FT response to exhaustive exercise between male cyclists supplemented with zinc sulfate (30 mg) for four weeks compared to a placebo-controlled group ^[53][160]. Although zinc has an important role in the regulation of testosterone production, long-term studies in competitive athletes have not been conducted. Whether zinc supplementation is effective only during periods of zinc deficiency or whether it can augment normal testosterone concentrations regardless of baseline concentrations is not well understood.

Magnesium is one of the most abundant minerals in the body. It has an important role in various biological systems including protein synthesis, cellular energy production, cell growth, and reproduction ^[54][161]. From an athletic performance perspective, magnesium is involved in skeletal muscle function and energy production, suggesting a possible ergogenic effect ^[55][162]. The recommended dietary allowance for magnesium intake for men is between 400 to 420 mg·day⁻¹ and 310 to 320 mg·day⁻¹ for women ^[56][163]. Several studies have reported that athletes do not consume enough magnesium from their diet, resulting in a greater risk for magnesium deficiency ^{[57][58][59][60]}[164,165,166,167]. Several investigations have reported a relationship between magnesium and testosterone concentrations ^[61][62][63][168,169,170]. One study indicated that magnesium supplementation in young healthy men in combination with a four-week endurance training program increased both FT and TT concentrations at rest and following exhaustive exercise ^[64][171]. An additional study conducted on nearly 400 older adult men reported a significant correlation between magnesium status and testosterone concentrations (r = 0.20, p < 0.05) ^[62][169]. The mechanism responsible for this relationship has yet to be elucidated. However, it is possible that it may be more indirect than direct. Magnesium is known to have a role in decreasing oxidative stress and inflammation ^[65][66][67][172,173,174]. Considering that testosterone concentrations can be strongly influenced by oxidative stress ^[68][175], it is possible that magnesium’s role in decreasing oxidative stress may provide the stimulus to maintain testosterone concentrations during periods of oxidative stress. A strong positive correlation has been reported between total antioxidative capacity and testosterone concentrations (r = 0.807) ^[68][175]. Magnesium has an important role in maintaining antioxidant capacity and controlling oxidative stress ^[65][66][67][172,173,174]. Magnesium deficiency has been demonstrated to increase production of oxygen free radicals, increase oxidative tissue damage, decrease antioxidant enzyme activity, decrease cellular antioxidant levels, and increase oxygen peroxide production ^[69][70][71][176,177,178]. In contrast, normal magnesium levels can prevent oxygen radical formation by removing free radicals and inhibiting xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase elevations ^[72][179].

Magnesium deficiency has also been associated with low-grade systemic inflammation ^[65][73][172,180], and has been shown to increase pro-inflammatory cytokines: tumor necrosis factor-alpha (TNF-α) and interleukin 1 (IL-1) ^[73][74][75][180,181,182]. Low-grade chronic inflammation has been shown to decrease testosterone concentrations by suppressing testosterone secretion from Leydig cells, resulting in both an inhibitory effect on LH secretion and reduced LH sensitivity at the Leydig cell ^[76][77][183,184]. Increases in TNF-α activates nuclear factor κB (NF-κB), a transcription factor that governs the expression of early-response genes involved in cellular responses to a wide range of signals ^[78][185]. NF-κB inhibits the activation of steroidogenic-enzyme genes such as Nur77 and SF-1, which regulate steroidogenesis (biosynthesis of testosterone from cholesterol) in the Leydig cells ^[77][184]. Rochelson and colleagues ^[79][186] demonstrated, through an in vitro examination, that magnesium sulfate can reduce the nuclear translocation of NF-κB. Others have demonstrated that magnesium supplementation can reduce inflammatory status and decrease levels of TNF-α and IL-1 ^[73][80][180,187].

Magnesium also appears to reduce the binding of testosterone to SHBG ^[81][188]. Most circulating testosterone is bound to SHBG; however, the bioavailability of testosterone is related to the free testosterone concentrations, which is only a fraction of circulating testosterone ^[82][189]. Magnesium appears to bind to SHBG resulting in the blocking of testosterone’s ability to bind to SHBG, subsequently enhancing testosterone bioavailability. Magnesium deficiency appears to increase testosterone binding to SHBG, potentially decreasing its bioavailability ^[81][188]. Whether magnesium supplementation is effective in augmenting testosterone synthesis as an anabolic agent is not well understood.