**1. Introduction**

In a normal body state, the naturally occurring free radicals in humans have been reported to exert significant positive effects on immune system regulation [1] and to have a significant negative effect, namely peroxidation, on lipids, proteins, and DNA [2,3]. Exercise has been demonstrated to enhance muscular metabolism, and consequently, oxygen uptake, which further enhances the generation of

free radicals and oxidative stress [4,5]. Apoptosis is a phenomenon that occurs when free radicals damage human DNA and cause accelerated programmed cell death [6]. One study indicated that the percentage of apoptotic cells increased significantly after running at an intensity of 80% maximal oxygen consumption (VO2max) until exhaustion, whereas it remained unchanged after running at an intensity of 60% VO2max for an identical running time [7]. Therefore, in athletes, the higher the exercise intensity is, the more apoptosis occurs. Adequate apoptosis is an essential mechanism in the human body [8], but a high percentage of apoptosis can induce alterations in the physiology and viability of circulating leucocytes, which have a causal relationship with exercise-induced immune distress [9]. Nutritional interventions to attenuate inflammation and apoptosis may directly or indirectly benefit muscular recovery and subsequent performance [10].

Betaine, a natural compound, is commercially obtained from sugar beet [11]. It was first discovered in *Beta vulgaris* in the 19th century [12] and has been noted to be present in microorganisms, plants, and animals [13], with wheat, shellfish, spinach, and beetroot containing high levels of the compound [14,15]. Betaine can not only be absorbed by the human body from diet but also be converted from choline. Choline, the precursor of betaine, can be oxidized to betaine aldehyde by choline dehydrogenase [12]. The betaine aldehyde can be oxidized to betaine by betaine aldehyde dehydrogenase in the presence of NAD<sup>+</sup> [16]. Therefore, the human body can also obtain betaine from foods rich in choline, such as eggs, meat, fish, and whole grains [17]. About 50% of choline in the intestine will be converted into betaine [18] and humans can obtain average 1 g of choline from daily diet [17]. As early as the 1990s, betaine was added to animal feed to evaluate its effects on growth performance [19,20] and disease prevention [21]. In the first study involving humans related to betaine supplementation and exercise performance, Armstrong et al. [22] observed that oxygen consumption during sprinting after acute betaine supplementation (5 g of betaine mixed in 1 L of carbohydrate–electrolyte fluid) was significantly higher than in those who consumed only carbohydrate–electrolyte fluid. However, betaine supplementation did not improve sprint performance in a hot environment. Furthermore, long-term betaine supplementation (1.25 g twice per day for 14 days) before an acute exercise session was noted to significantly increase the concentrations of growth hormone and insulin-like growth factor-1 as well as significantly decrease cortisol concentration [23]. Notably, a 14-day betaine supplementation was suggested to effectively promote protein synthesis. However, other studies have revealed that long-term betaine supplementation had no benefits on jump squat power, the number of bench press or squat repetitions [24], or the peak concentric or eccentric force outputs during isokinetic chest press [25]. Therefore, the effects of betaine supplementation on exercise performance remain unclear.

In addition to improving strength and power, betaine regulates organic osmolytes and protects the function of cells and mitochondria [12]. Therefore, some cell culture and animal studies have investigated the effects of betaine supplementation on apoptosis and oxidative stress in damaged cells [26–29]. A study by Veskovic et al. [26] observed that betaine decreased the liver's expression of proapoptotic mediator Bax and increased antiapoptotic Bcl-2 in nonalcoholic fatty liver disease induced by a methionine–choline-deficient diet in mice. In addition, betaine increased superoxide-dismutase, catalase, glutathione peroxidase, and paraoxonase activities. Studies have suggested that betaine can effectively attenuate apoptosis and improve antioxidative defense. In addition, other studies have determined that betaine exerts antiapoptotic effects in human corneal epithelial cells [27] and antioxidative stress effects in the liver of rats [28,29]. Furthermore, our laboratory data revealed that a single dose of betaine supplement (1.25 g of betaine mixed in 300 mL of sports beverage) 1 h before an exhaustive endurance exercise (EEE) significantly decreased lymphocyte apoptosis but had no effects on mitochondrial transmembrane potential (MTP) decline [30].

Based on these aforementioned results, we hypothesized that long-term betaine supplementation attenuates apoptosis and oxidative stress induced by exercise and enhances aerobic capacity. Therefore, this study evaluated the effects of 2 weeks of betaine supplementation on apoptosis, oxidative stress, and aerobic capacity after EEE.
