Spirulina is the other microalga approved for human consumption [4]. Preparations of chlorella and spirulina have different proportions of common components. The polysaccharide (β-glucans and arabinomannans) composition of Chlorella spp. has been better defined than that of Spirulina [4]. Chlorella contains β-glucans (polymers of β-D-glucose linked through 1–3 β-glycosidic bonds, 6–9% of dry weight) [8] and arabinomannans (oligomers of arabinose and mannose) [9] as well as other less known saccharides with hypolipidemic activity [10]. As well as these lipidemic functions, up to 50% of Chlorella dry weight can consist of triacylglycerols (TAGs) when it has been exposed to high light or nitrogen deficiency conditions [11]. Peptides from the enzymatic hydrolysis of Chlorella can also have health benefits, such as hypoglycemic, anti-inflammatory, and blood pressure-lowering activities (e.g., inhibition of angiotensin I-converting enzyme, ACE) ^[4][12][13][4,12,13]. Therefore, because of its nutritional value and bioactive compounds, Chlorella is an interesting microalga for human consumption; however, if it is not properly processed, it could be poorly digested and induce side effects because, in its naturally occurring form it contains a cellulose cell wall. So, chlorella must be mechanically broken down during production for human consumption to avoid gastrointestinal issues, including nausea, vomiting, and stomach problems. Other side effects related to chlorella intake include renal and allergy problems [14]. Despite this, in the context of human health and disease, chlorella has been shown to have cardiovascular benefits, as revealed by its capacity to lower total cholesterol, low-density lipoprotein cholesterol, systolic blood pressure, diastolic blood pressure, and fasting blood glucose in healthy individuals and patients with non-alcoholic fatty liver disease, hypertension, hypercholesterolemia, and dyslipidemia [15]. At the doses used to elicit these responses, chlorella is considered to be safe [16].

In addition to its macronutrients, chlorella also contains phenolic compounds with radical scavenging capacity and α-amylase/glycosidase inhibitory activity [12], as well as other components such as vitamins (e.g., vitamin B₉ or folate) [17] and carotenoids (e.g., astaxanthin) [18] that may contribute to its antioxidant and anti-inflammatory activities [19]). Moreover, it incorporates chlorophyll and other minerals with some biological functions [7]. Taken together, it seems that the functional activity of chlorella intake could be complementary or synergistic with other health-related habits, such as consuming a balanced diet, getting adequate sleep, or taking physical exercise.

Physical exercise represents a stressful activity for the body’s cells, tissues, and organs, which dysregulates whole-body homeostasis in a progressive and reversible way. However, as a consequence of its regular and systematic practice, different cellular signaling pathways become activated and generate systemic and local chronic adaptations. These promote physiological, biochemical, and morphological adjustments of the organism to the exercise [20], such as a gain of muscle mass and strength and improvements in cardiovascular health and functional capacity [21].

Besides their role as biochemical triggers and mechanical platforms for strength production, skeletal muscle fibers are nowadays also considered to be essential pleotropic sources of several compounds and molecules crucial for muscle-to-organ/tissue cross-talk communication [22]. Myokines, for example, are specific cytokines, small peptides, growth factors, and metallopeptidases produced and released by skeletal muscle, with both paracrine and endocrine effects, that promote communication between muscle cells and other target organs or tissue cells [21]. It has been shown that regular exercise increases the production and secretion of myokines, such as irisin, insulin-like growth factor 1 (IGF-1), brain-derived neurotrophic factor (BDNF), meteorin-like protein (Metrnl), fibroblast growth factor, β-aminoisobutyric acid (BAIBA), the interleukins (ILs) IL-15, IL-7, IL-6, and decorin, whilst reducing the production and release of myostatin. These myokines are the drivers of the most beneficial effects of exercise in terms of health-related outcomes and chronic adaptive responses to training ^[23][24][23,24]. This is because their regulation is directly related, among other things, to increased protein synthesis and reduced muscle protein breakdown, increased lipid oxidation, browning of white adipose tissue, increased insulin sensitivity, increased myogenesis, and satellite cell activation (for a review, see [25]). Furthermore, it is known that, both during and after conventional exercise models, contracting muscles increase ROS production in a way that positively impacts muscle cellular signaling and adaptive responses, thereby strengthening cells to deal better with the demands of physical exercise and to mitigate several deleterious consequences associated with pathological conditions ^[20][22][20,22]. Regarding health-related benefits, regular exercise has been defined as a prophylactic but also a therapeutic, non-pharmacological “polypill”, able to prevent around 26 different chronic diseases and reduce all-cause mortality ^[23][24][26][23,24,26], by reducing the incidence of cardiovascular diseases; through the regulation of blood lipids, hypertension, and arterial fitness [22]; and by protecting against atherosclerosis, type 2 diabetes, and breast and colon cancer [23]. Moreover, regular exercise is a consensual treatment tool against different pathological risk factors or situations, such as obesity (by helping practitioners to lose weight, reducing the metabolic risk factor, and improving adipose tissue health ^[22][27][22,27]), ischemic heart disease, heart failure, type 2 diabetes, chronic obstructive pulmonary disease, chronic low-grade systemic inflammation, and non-alcoholic fatty liver disease [23]. Furthermore, physical exercise has been considered crucial to mental health, helping in the management of depression and anxiety and in the therapeutic challenge against neurodegenerative disorders such as Alzheimer’s or Parkinson’s disease ^[28][29][28,29]. Apart from preventing and treating pathological situations, regular physical exercise can improve cardiovascular fitness and aerobic capacity, thereby slowing biological aging through a reduction of age-related sarcopenia and thus increasing quality of life and potentially life expectancy.

Chlorella, as well as spirulina and other microalgae, is progressively being introduced into human and animal nutrition as a wholesome source of nutrients that is complementary or an alternative to animal-derived foodstuffs. Whereas research on the properties of edible microalgae in the context of health and disease has been summarized many times, the important area of sports nutrition has not been systematically reviewed to date. Therefore, the present rpapesearchr aims to contribute to the advancement of research on the use of new environmentally friendly and health-promoting foodstuffs in this area. Taken together, it is reasonable to assume that microalgae and their bioactive compounds are interesting nutritional sources to improve exercise performance and post-exercise recovery, as well as enhance the beneficial clinical features of physical exercise [1].

2. Bioactive Macronutrients from Chlorella and Physical Exercise

2.1. Benefits of Proteins and Peptides from Chlorella for Physical Exercise

Chlorella species have a great potential to be used as an alternative protein source because over 80% of them are digestible by humans and comparable both quantitatively and qualitatively to other conventional vegetable proteins ^[30][49]. In terms of quantity, proteins range from 51% to 58% in C. vulgaris, and 57% for C. pyrenoidosa on a dry weight basis in growth conditions that are rich in nitrogen. In the absence of nitrogen, the amount of proteins present can decrease by up to 20% w/w on a dry weight basis ^[30][31][32][49,50,51]. The current trend of increasing atmospheric CO₂ may also lead to an increase in the protein content of these species ^[32][51]. In terms of quality, most microalgae contain all of the essential amino acids that mammals are unable to synthesize and therefore must obtain from their food intake ^[31][50]. Specifically, the essential amino acid index (EAAI) of C. pyrenoidosa indicates that all essential amino acids are present at substantial concentrations ^[33][52]. Compared to other macronutrients, protein intake increases satiety, activation of the anabolic mTOR (mammalian target of rapamycin) pathway in skeletal muscle, and the thermic effects of meals ^[34][53]. As plant-based diets seem to be a good option for enhancing athletic performance while also improving general physical and environmental health ^[35][54], a regular intake of Chlorella or its isolated proteins could be an environmentally friendly choice of dietary supplement for people undergoing training. Apart from its protein content, Chlorella also contains bioactive peptides that may be of interest in terms of exercise performance. Bioactive peptides usually contain 3–16 amino acid residues, and their activities are mainly based on their amino acid composition and sequence ^[36][55]. Some bioactive peptides are antioxidants, as they scavenge ROS and free radicals or prevent oxidative damage by interrupting the radical chain reaction of oxidation. This antioxidant activity of peptides mainly depends on the hydrophobic amino acids they contain, specifically some aromatic amino acids and their histidine content ^[37][56]. A peptide from C. ellipsoidea (LNGDVW, 702.2 Da) has demonstrated great efficiency in scavenging various free radicals in vitro and therefore has the potential to be a good dietary supplement for the prevention of oxidative stress ^[38][57]. Another peptide from C. vulgaris (VECYGPNRPQF, 1309 Da) can efficiently quench a variety of free radicals, including hydroxyl, superoxide, peroxyl, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, and also has significant protective effects on DNA and against cellular damage caused by hydroxyl radicals ^[39][58]. So, the intake of certain peptides from Chlorella with a tested antioxidant capacity could improve the detrimental oxidative stress potentially induced by particular high-intensity bouts of exercise or periods of exacerbated exercise regimens and training schedules [40]. Most analyses show that the highest proportions of amino acids in marine algae, and specifically in most Chlorella species, are glutamic and aspartic acids ^[6][41][6,59]. Besides the organoleptic properties of glutamate (one of the main components of the savory flavor, contributing to the basic umami taste), it also has bioactive activities that are potentially interesting in the context of exercise. Dietary supplementation with glutamic acid increases intramuscular free amino acid (FAA) concentrations and decreases the mRNA levels of genes involved in protein degradation in the skeletal muscle of growing pigs ^[42][60]. Moreover, when combined with leucine, it improves the FAA profile and mRNA levels of amino acid transporters in muscle, including neutral amino acid transporter 2 (ASCT2), large neutral amino acid transporter (LAT1), sodium-coupled neutral amino acid transporter 2 (SNAT2), low-affinity intestinal transporter of glycine and imino acids (PAT-1), and high-affinity renal transporter of glycine, proline, and hydroxyproline (PAT2) ^[43][61]. Meanwhile, aspartate, the other main amino acid in chlorella, enhances the ability of muscle to utilize free fatty acids during moderate exercise, thereby sparing glycogen and improving the biochemical capacity of the muscle for the oxidation of fatty acids through β-oxidation ^[44][62]. Chlorella products also contain high levels of arginine, a pivotal amino acid for the production of NO and the regulation of the immune system [7]. L-arginine supplementation in combination with other components improves tolerance of aerobic and anaerobic physical exercise in untrained and moderately trained subjects ^[45][63]. Moreover, supplementation with L-arginine together with physical training seems to be an important stimulus that induces significant improvements in exercise performance and redox status in rats ^[46][64]. Numerous papers have provided evidence that Chlorella-derived bioactive peptides have notably beneficial effects on human health. This evidence suggests that various amino acids and their metabolites play important roles in the skeletal muscle and have a significant overall ergogenic effect on exercise.

2.2. Benefits of Polysaccharides from Chlorella for Physical Exercise

A key part of the bioactivity of chlorella might be associated with its polysaccharides ^[47][65]. Polysaccharides with different structural features from various Chlorella species present a variety of biological activities, such as immunomodulatory, antioxidant, hypolipidemic, antitumor, or anti-asthmatic ^[48][66]. Research into the bioactivity of polysaccharides extracted from C. pyrenoidosa has focused on their effects on immunity and their antioxidant activities ^[48][66]. The polysaccharide fractions from C. pyrenoidosa, consisting mainly of the d-arabinose, d-glucose, d-xylose, d-galactose, d-mannose, and l-rhamnose moieties, have in vitro antitumor ^[49][67] and antioxidant capacity, specifically against superoxide and hydroxyl radicals ^[48][66]. α and β-Glucans, polysaccharides present in C. pyrenoidosa and C. sorokiniana ^[47][65], can modulate ROS production, the expression of the ROS-generating enzyme dual oxidase 2 (DUOX-2), and the immune factors TNF-α, IL-1β, and COX-2, thereby contributing to maintaining low levels of oxidative stress and pro-inflammatory molecules ^[50][68]. Dietary fiber consists of complex polysaccharides that are poorly or non-digestible by humans. High-fiber diets improve glycemic control, blood lipids, body weight, and inflammation ^[51][69]. C. vulgaris contains dietary fiber in highly variable proportions, ranging from 5.6% to 26.0% w/w on a dry weight basis ^[52][70]. In Chlorella-derived products, more than 65% of their carbohydrate content is dietary fiber from the Chlorella cell wall [7]. Soluble dietary fiber is mainly composed of resistant or functional oligosaccharides, such as fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), and inulin, and viscous dietary fibers with a high molecular weight (glucan, pectins, and gums) ^[53][71]. Several studies suggest that both resistant oligosaccharides and viscous dietary fibers can effectively increase the bacterial diversity of the human gut microbiota and the abundance of bifidobacteria, lactobacilli, Prevotellaceae, and Faecalibaculum spp. ^[54][72]. Recent research has revealed a connection between the profile and diversity of the gut microbiota and the host’s physical performance ^[55][73]. Fiber intake promotes gut microbial diversity and increases the proportion of species, such as Faecalibacterium prausnitzii, Lactobacillus spp., Bifidoacterium spp., Firmicutes, and Bacteroidetes, that produce SCFAs ^[56][74]. SCFAs, including acetate, butyrate, and propionate, exert a wide range of metabolic functions, including anabolic regulation, insulin sensitivity, and modulation of inflammation, when absorbed into systemic circulation ^[57][75]. In vitro, C. pyrenoidosa increases the abundance of the genera Prevotella, Ruminococcus, and Faecalibacterium, as well as the production of butyrate and propionate ^[58][76]. Meanwhile, C. pyrenoidosa polysaccharides (CPPs) reduce Escherichia-Shigella, Fusobacterium, and Klebsiella and increase Parabacteroides, Phascolarctobacterium, and Bacteroides ^[59][77]. At the phylum level, CPPs increase the abundance of Bacteroidetes, leading to a lower Firmicutes/Bacteroidetes ratio ^[59][77], which seems to be closely related to body weight control and to the maintenance of intestinal barrier integrity, as precursors of low-grade inflammation, which are both important aspects affecting exercise performance. Other Chlorella species, such as C. vulgaris and C. protothecoides, increase propionate-producing bacteria in vitro ^[60][78]. In rats, a CPP treatment increased the growth of Coprococcus, Turicibacter, and Lactobacillus and the concentrations of acetate, propionate, and butyrate ^[10][61][10,79]. C. vulgaris also modulates Lactobacillus spp. metabolism, thereby increasing growth and the production of l-lactic acid while reducing the production of d-lactic acid ^[61][79]. Increasing the proportion of Lactobacillus plantarum in the gut seems to augment muscle mass, enhance energy harvesting, and have health promotion, performance improvement, and anti-fatigue effects ^[62][80]. Other aspects of physical performance can also be directly improved by SCFAs ^[55][73]. SCFAs can be used as carbon and energy sources for liver and muscle cells, thus improving endurance performance by maintaining blood glucose levels ^[63][81]. SCFAs also appear to regulate neutrophil function and migration, inhibit inflammatory cytokines, and control the redox environment in cells, which may help to enhance muscle renewal and adaptability, improve exercise performance, and delay symptoms of fatigue ^[63][81]. However, the efficacy of chlorella supplementation depends on the gut microbiota of the host ^[64][82], so future studies should focus on the underlying mechanisms implicated in the crosstalk between Chlorella polysaccharides, gut microbiota, and exercise performance in order to maximize the metabolic benefits of polysaccharides from Chlorella.

2.3. Benefits of Fatty Acids from Chlorella for Physical Exercise

Chlorella synthesizes high levels of unsaturated fatty acids in response to some environmental factors, such as temperature, pH, light, air composition (mainly nitrogen and phosphorous limitations), salinity, and nutrients ^[65][83]. The lipid composition of C. vulgaris includes about 23–34% of saturated fatty acids (SFAs), 15–25% of monounsaturated fatty acids (MUFAs), and 42–62% of PUFAs (of which over 30% are ω3 PUFAs) by weight as a percentage of the unfractionated total lipids ^[66][67][84,85]. The major PUFAs in C. vulgaris are linoleic acid (LA, C18:2 ω6; over 23%) and alpha-linolenic acid (ALA, C18:3 ω3; over 21%) ^[66][84]. LA is considered an essential fatty acid because higher animals, including humans, cannot synthesize it ^[68][86]. The proportions of total unsaturated fatty acids and PUFAs in C. vulgaris are the highest of the green microalgae ^[66][84], and they can be increased under favorable growing conditions. This makes it suitable for industrial and nutritional purposes ^[69][87]. Incorporating specific nutrients or dietary supplements to enhance exercise performance and recovery is a common strategy used by recreational practitioners and, particularly, by professional athletes. Moreover, supplementation with unsaturated fatty acids has been shown to have a number of biological effects on health and is related to diseases ^[70][88]. Many studies have evaluated the impact of unsaturated fatty acid supplementation, mainly with ω3 PUFAs, on exercise performance, because of their antioxidant effects ^[71][89]. As mentioned before, particular conditions of physical exercise, specifically those involving extremely intense exercise, very concentrated training, or periods of competition, as well as exercise sessions with a high predominance or proportion of eccentric contractions, can result in increased oxidative damage to cellular constituents. This is because such exercise increases the production of ROS/RNS in skeletal muscle above a physiological threshold ^[72][90]. Under these conditions, instead of being positive triggers of adaptive cellular signaling pathways, high levels of ROS cause functional oxidative damage to proteins, lipids, and other cell components that could exacerbate atrophy, sarcopenia, and myopathy factors in muscle. The persistence of greatly elevated levels of ROS at the local level may also reduce muscle reparation and differentiation of myoblasts and myotubes ^[73][91]. However, it has been shown that the MUFAs in chlorella reduce lipid peroxidation and oxidative stress damage ^[70][88]. Additionally, ω3 ALA is an antioxidant fatty acid that enhances eNOS activity and inhibits superoxide and peroxynitrite formation, thereby contributing to endothelial protection among other possible activities ^[74][92]. The effect of ω3 ALA on endothelial function can also be related to its activity on SIRT3 impairment, which in turn restores the mitochondrial redox balance in endothelial cells ^[75][93]. The antioxidant potential and free radical scavenging activity of ω3 ALA can also protect against cellular damage, apoptosis, and inflammation ^[76][77][94,95]. Inflammatory responses have been observed in athletes who engage in long-duration exercise, such as marathons or triathlons ^[78][96]. Inflammation is a physiological response to tissue damage that increases the expression of TNF-α, IL-1β, and IL-6 via NFķB. After muscle injury, NFķB increases the expression of RING-finger protein-1 (MuRF1), eventually promoting muscle wasting ^[79][80][97,98]. ω3 ALA intake seems to reduce plasma concentrations of IL-6 and other molecules related to inflammatory signaling, such as C-reactive protein, E-selectin, ICAM-1, and soluble vascular cell adhesion molecule 1 (VCAM-1), thereby helping to control the inflammatory response ^[81][99]. Several epidemiological studies show that ω6 LA also reduces the inflammatory response, despite its pro-inflammatory effect in vitro ^[82][100]. An adequate proportion of these two principal PUFAs (ω6 LA and ω3 ALA) in chlorella seems important to arrive at the desired anti-inflammatory effect, as a high ω6 diet can inhibit the anti-inflammatory and inflammation-resolving effects of the ω3 fatty acids ^[82][100]. A reduction of plasma IL-6 by ω3 ALA has also been reported during resistance training in older men ^[83][101]. This action may be based on the stimulation of muscle protein synthesis and enhancement of muscle mass via mTOR signaling, as described for ω3 PUFAs ^[84][102]. Insulin signaling plays a key role in mTOR activation, so PUFA supplementation might alleviate anabolic resistance ^[85][103] and improve the action of insulin, as skeletal muscle is the major site of insulin-stimulated glucose disposal ^[86][104]. Interestingly, metabolic benefits in terms of muscle glycemia have been reported for ω6 LA rather than ω3 PUFA ^[87][105], and there is an inverse association between the serum proportion ω6 LA and both fasting plasma glucose and post-load glucose ^[88][106]. ω3 ALA is also active at the metabolic level, as it increases the activity of carnitine palmitoyl transferase I and FA translocase ^[89][107]. The activity of these enzymes increases maximal fat oxidation in mitochondria, thus supporting the assumption that ω3 ALA intake may be used as a nutritional support to increase aerobic performance with substantial reliance on lipid-based metabolism and, at the same time, a glycogen sparing effect ^[89][107]. Dietary ω3 ALA and ω6 LA are oxidized much more rapidly than other biologically active fatty acids, such as docosahexaenoic acid (DHA) and arachidonic acid ^[90][108]. Specifically, ω3 ALA has been shown to have the highest rate of β-oxidation among the unsaturated fatty acids tested ^[68][86], so ω3 ALA could serve better than other fatty acids, as an energy substrate during long-term exercise bouts when carbohydrate reserves are depleted. The dietary lipid profile determines the tissue phospholipid composition, which modulates not only insulin signaling but also the fluidity and rigidity of the cell membrane ^[91][92][109,110]. The administration of ω3 ALA maintains the integrity of the membrane in red blood cells (RBCs) when exposed to oxidative damage, and reduces lipid peroxidation ^[77][95]. The inclusion of ω3 PUFAs in the phospholipids of the RBC membrane increases the loss of deformability induced by exercise, thus improving performance by enhancing oxygen transport to the skeletal muscle ^[93][111]. However, most studies of ω3 PUFAs and RBC deformability have used fish oil, which is rich in ALA-derived ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), but not with the major fatty acids found in Chlorella (LA and ALA). Additionally, there is some concern about the adequate dosage of ω3 fatty acids, as excessive incorporation into plasma and tissue lipids may increase their susceptibility to lipid peroxidation, which is much more evident in athletes who may undergo high levels of oxidative stress ^[93][111]. So, further studies are still needed to provide better knowledge of the effects of fatty acids from Chlorella on cell membrane dynamics and the implications in the context of physical exercise.