Intense exercise may cause acidosis leading to ATP depletion. At the beginning of the exercise, ADP is converted to ATP. As the exercise increases in intensity and duration, the ATP:ADP ratio decreases due to ATP consumption, and the total adenine nucleotide pool decreases due to adenosine monophosphate (AMP) deamination. To maintain the ATP:ADP ratio, energy-rich phosphate groups are transferred from one ADP to another, each time resulting in one ATP and one AMP. Prolonged or intense exercise can activate AMP-kinase, a key sensor of cellular energy stress involved in long-term metabolic adaptations, such as the increase in the number of mitochondria. Because AMP degradation is enhanced during exercise, serum ammonia and intracellular inosine monophosphate (IMP) levels concomitantly increase. The progressive accumulation of the deaminated products IMP and ammonia cause fatigue. This explains, for example, the lower fatigue tolerance in patients with AMP deaminase deficiency [35]. Monitoring acidosis may thus give an idea of muscle fatigue since the decreased ATP activity affects the ryanodine receptor function and impairs the functioning of ATPases pumps in several myocytes [15, 35].

Acidosis leads to a shift in ATP production from aerobic to anaerobic processes, thus producing lactate. Hypocalcemia and hypomagnesaemia may follow this condition and are easily detected in blood tests. Since ammonia closely follows the lactate response during exercise, it lends itself to monitoring muscle fatigue. In clinical practice, the ischemic test (a forearm exercise under ischemic conditions in a fasted subject) can easily reflect acidosis condition, enabling to easily obtain the dosage of serum lactate and ammonium, prior to, during, and after the test. Likewise, purine derivatives hypoxanthine and xanthine are increased in serum and urine after prolonged exercise. Since they are directly correlated with the amount of ATP consumed inside the cell, they are considered good biomarkers of muscle fatigue [15].

Neuro-inflammation is an important factor that causes and follows motor neuron damage, muscle degeneration, and the occurrence of neurological fatigue. High levels of oxidative stress biomarkers and a reduction of antioxidant factors are observed in ALS patients [26, 27]. For instance, the nuclear factor erythroid 2-related factor 2 (Nrf2) protects against oxidative stress and cell death induced by the SOD1-mutant protein and increases the resistance of glial cells to oxidative stress by increasing glutathione (GSH) levels in SOD1-mutant protein mice [36]. Studies in vitro found that motor neuronal cell lines expressing TDP-43 mutants exhibited shortened neuritis and higher oxidative stress, an effect partially reversed using the Nrf2 enhancer. Thus, all markers of motor neuron injuries may give an indirect measure of muscle fatigue. For instance, markers like reactive oxygen species (ROS) and reactive nitrogen species (RNS), cytokines like TNF, interleukin 1b (IL-1b), IL-10, IL-4, IL-6, chemokine, and brain-derived neurotrophic factor (BDNF) are easily measured in blood, giving a picture of inflammatory state [26, 36].

Under physiological conditions, ROS are required to control biochemical processes, including cell differentiation, neurogenesis, antioxidant gene expression, and immune system regulation [37, 38]. However, conditions of muscular stress, including muscular inflammation related to some types of muscular exercise, may increase oxidative stress, thus leading to an oxidation of proteins, lipids, or nucleic acids at the basis of signaling pathways inducing necrosis and apoptosis [38]. In healthy subjects, muscle fibers can prevent oxidative damage, so that during training reproducing a muscular stress condition, the total antioxidant capacity (TAC) shows an increase at the beginning of the exercise and after high-volume training. This suggests that the antioxidant defense system is activated during exercise. Muscle cells are able to create a protective environment against oxidative stress, recruiting anti-inflammatory molecules like IL-6, IL-8, IL-15, BDNF, fibroblast growth factor 21, and follistatin-like-1 [15, 38].

ROS can activate transcription factors known to regulate IL-6, a cytokine acting both as pro-inflammatory (in monocytes and macrophages), and as anti-inflammatory (in myocytes), which is easily detectable in blood in conditions of increased inflammation [38]. However, in conditions of neurodegenerative disorders, the increased oxidative stress can exceed the muscular antioxidant capacity [15, 38], making ROS and TAC potential biomarkers. The dosage of ROS and TAC are easily measured in a blood test, and their dosage coupled with biomarkers of oxidative damage [such as lipid peroxidation biomarkers like thiobarbituric acid-reactive substances (TBARS), and isoprostane, protein oxidation biomarkers–advanced oxidation protein products (AOPPs) or biomarkers of antioxidant capacity], may give a more accurate picture of muscle fatigue [15]. While ROS immediately peaks after exercise, TBARS significantly decreases post-exercise, likely in relation to the time required to activate the biological pathways underlying the cellular redox state after an aerobic exercise [39].

In addition to TAC, GSH and glutathione peroxidase (GPX) are easily tested in serum and saliva. They are scavengers of ROS and hydrogen peroxide (H₂O₂), respectively, and the former increases with high-volume training, while the latter is able to scavenge at low-volume training. At higher exercise workloads, the production of H₂O₂ exceeds GSH and GPX capabilities, leading to a loss of muscle contractility, and to an increase in muscle fatigue [15, 40].

Concerning the redox balance and its relationship with exercise in ALS patients, Pasquinelli et al. [41] measured oxidative parameters in ALS patients during an exercise protocol using a myometer at a forearm in which the contractile force increased incrementally. Plasmatic levels of AOPPs, Ferric reducing antioxidant power (FRAP), and total thiol (t-SH) groups remained stable during the short-lasting exercise protocol, suggesting a lack or delayed exercise-related kinetic curve for these redox biomarkers in ALS patients. On the contrary, levels of lactate during each step of the exercise protocol increased similarly in ALS patients compared to healthy controls. Interestingly, they found different results in patients carrying different polymorphisms in the PGC-1α gene, which is activated by muscle contraction and brings fast-to-slow muscle fiber conversion. Patients harboring the Ser428Ser genotype showed higher levels of AOPPs [at 50% of maximal voluntary contraction (MVC) force and at recovery] and lactate (at 30% and 50%) during exercise compared to Gly482Gly patients [41].

IL-6 can function as a myokine, increasing in response to muscle contractions [42]. It increases with exercise duration, reaching the maximum levels immediately post-exercise, and returning to resting levels a few hours after the exercise ends [15]. During exercise, IL-6 may act in a hormone-like manner to mobilize extracellular substrates or augment substrate delivery. It expresses its anti-inflammatory role by inhibiting TNF-α and IL-1 and activating IL-1RA and IL-10. TNF-α is a pro-inflammatory cytokine able to induce apoptosis and inflammation [26, 42].

Finally, since the neutrophil-to-lymphocyte ratio is reported to be correlated to the degree of neuroinflammation in ALS patients [43], blood cell count may also be considered to add information on inflammation.

Once fatigue is detected and quantified, there is the need to reduce its symptoms by therapeutic means. Although no specific treatments for fatigue in ALS exist, several therapeutic options are available.

According to a recent Cochrane review [10], modafinil showed significant results in clinical trials testing clinical outcomes in regard to FSS and to measures of depression and sleepiness. Modafinil acts by stimulating the release of norepinephrine and dopamine from synaptic terminals, thus elevating hypothalamic levels of histamine. Creatine, which increases the maximum availability of energy output in anaerobic activities, may also have a positive effect on muscle strength and fatigue. Preliminary studies showed that natural molecules such as curcumin, a natural antioxidant compound, might reduce oxidative stress, and be combined with disease-modifying therapies [44]. Among non-pharmacological approaches, resistance exercise like treadmill ambulation and muscular exercise showed a reduction in fatigue at FSS. Repetitive TMS (rTMS) is a well-established therapeutic strategy that, by increasing cortical plasticity, may be used to boost motor rehabilitation and treat mood disorders [43, 45]. In a clinical trial, the patients group performing rTMS at 5Hz on the motor cortex for 2 weeks showed a reduction in FSS versus the control group which performed a sham intervention [46]. Future applications of nanomaterials bear promise towards the treatment of ALS fatigue. The increasing feasibility of tissue-specific delivery of oxygen using nanobubbles makes it a promising approach to compensate for hypoxia, a potential complement to current therapeutic approaches [47]. Although still in a preliminary testing phase, antioxidant materials coupled with carbon nanotubules have been shown to be able to support the regeneration of neuronal stem tissues [48] and the reduction of oxidative damage [49]. Similarly, selenium-based nanoparticles have recently shown neuroprotective effects on other neurological disorders as well [50].

Besides pharmaceutical and technologic approaches to therapy, the role of physical exercise has been debated for a long time. In the past, the main idea was to avoid physical activity since it may increase muscular consumption. In the last years, however, more and more studies pointed out that short-lasting muscular training that does not reach a threshold of muscular fatigue is considered to be beneficial for ALS patients [45]. However, other reasons for uncertainty in promoting sports in ALS derive from previous studies that associated strenuous exercise with higher ALS incidence, as observed in athletes and professional football and American football players [6, 51]. According to this interpretation, an intense and prolonged exercise may induce a massive increase in ROS and calcium concentration, with consequent motor neuron degeneration [38].

In predisposed patients, very intense physical activity may simultaneously act on changing cortical plasticity in the motor cortex and in stressing muscles. The role of physical exercise in ALS was recently pointed out in a work by Julian et al. [52] that demonstrated that ALS shares polygenic risk genetic factors with several conditions, among which moderate to high levels of exercise. Transcriptomic analysis corroborated this result, revealing that genes whose expression resulted altered in acute exercise were enriched with known ALS risk genes (including C9ORF72), and with ALS-associated variants of unknown significance [6].

Since intense physical exercise can lead to an increase in oxidative stress, ALS patients seeking exercise as a treatment for fatigue must be followed by a physiotherapist with an experience in neuromuscular diseases, able to establish the correct fatigue threshold and the correct amount of muscular exercise [51]. In this view, biomarkers may help in defining this threshold, and in defining a physical program tailored to the patients’ clinical picture, since the spectrum of motor neuron diseases is widely heterogeneous and the muscular involvement is different. Although intense and excessive physical exercise may promote oxidative stress, moderate and routine exercise is associated with numerous benefits on cardiovascular, endocrine, and even neuromuscular factors. Adequate exercise training can enhance endogenous antioxidant defense systems, while intense and strenuous exercise may lead to ROS overproduction [38].

It can be assumed that physical exercise can have positive or negative effects on oxidative stress depending on the load, specificity, and basal level of training [15]. In previous studies, Siciliano et al. [38] tried to understand if exercise might be considered as a therapeutic strategy in neuromuscular diseases, and Zhu et al. [53] conducted a meta-analysis to establish which exercise better improved respiratory function, fatigue, and quality of life in ALS patients [38, 53]. These studies showed that intense aerobic exercise increases oxygen consumption with a consequent rise in ROS production, while for low exercise intensity [< 50% of maximal oxygen uptake], better antioxidant activity is present. After exercise, positive changes in redox status appear several hours after the exercise ends, likely following the time required for antioxidant gene transcription activation, mRNA maturation, and mRNA translation into protein. Thus, aerobic exercise should be prolonged in time and performed routinely [38].

Referring to anaerobic exercise, data are more controversial. The muscular exercise that creates a condition of hypoxia in muscle cells, like in the case of some type of anaerobic exercises, produces an increase in xanthine oxidase (XO), an enzyme that generates ROS during ischemia-reperfusion. Elevated lactate levels, an alteration of the oxidative status, an increase in the lipid peroxide concentration, and a decreased SOD activity were all found to occur in anaerobic exercise. However, the picture is arguably more complex, and other studies found an increase in antioxidant enzymes. Also in the case of anaerobic training, moderate-intensity training rather than maximal exercise may be able to improve adaptive responses to oxidative stress [38, 54]. In ALS-mice models, aerobic training led to an extension in the lifespan of SOD1-mutated mice, which showed higher levels of antioxidant activity. Mitochondria in trained ALS-mice models showed a reduction in lipid peroxidation compared to those in ALS-mice devoid of training. In the same study, ALS mice subjected to swimming or to running on a treadmill showed delayed spinal motor neuron death and preserved larger motor neurons. In contrast, mice forced to high-intensity training showed motor neuron loss and an increased proportion of motor neurons with small soma areas. Authors of these studies argued that running was a high-impact exercise, which recruited only small motor neurons and generated more oxidative stress than swimming, which was a low-impact exercise that recruited both small and large motor neurons [55]. On this same line, endurance exercises may protect skeletal muscle against the excessive activation of autophagy and ubiquitin-proteasome system, upregulating mitochondrial metabolism and preventing fiber-type transformation [56].

Although ALS-mice models employed in therapeutic trials may show encouraging results, findings may eventually not be confirmed in the following phases on patients due to the inherent complexity of the interrelation between muscular functions and structures. Still, mice models may disclose useful insights into the effects of pharmacological and non-pharmacological therapeutic approaches that are later confirmed in humans and may eventually lead to changes in therapeutic approaches and outcomes. Several studies on humans confirmed these findings and showed that physical activity led to an improvement in respiratory function [57], on ALS functional rating scale (ALSFRS) [58], and a reduced decline in leg strength [59]. Other studies did not confirm these beneficial effects [60, 61], though an improvement in mood and in quality of life was still often detected. The meta-analysis from Zhu et al. [53] revealed that combined programs of aerobic exercise, resistance exercise, and standard rehabilitation were the best in improving quality of life and in reducing fatigue in ALS patients, while exercise programs of aerobic and resistance training showed the highest potential to improve ALS patients’ physical function. Overall, literature findings mostly agree in supporting resistance exercise programs as a non-pharmacological adjuvant therapeutic approach in ALS [38, 53].