Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder. The only established epidemiological risk factors for ALS are male sex and increasing age. The role of physical activity has been debated as an environmental risk factor. Over the last decade multiple studies have attempted to delineate the architecture of ALS. These have not yet established definite risk factors, often due to low-powered studies, lack of focus on at-risk genotypes and sub-optimal methodology.n = 93), highlighting the underlying reasons for any reported discrepancies.We have conducted a review of all the studies published between 2009 and December 2021. The free text search terms were [(motor neuron disease) OR (MND) OR  OR (ALS)] AND [(Exercise) or  or (PA) or (sport)]. We identified common themes, for example soccer, head injury and the physiological mechanisms that differ in ALS patients. We have analysed the relevant, available studies (Overall, we have found that the more highly powered studies using validated exposure methodologies, linked strenuous, anaerobic physical activity as a risk factor for ALS. Future large-scale studies focusing on specific at-risk genotypes and physical activity should be conducted to confirm this finding. This will strengthen the evidence already surrounding strenuous physical activity as an environmental risk factor for ALS and allow advice to be given to at-risk family members. Increasing our understanding of the genetic–environmental interactions in the pathophysiology of ALS will allow for the possibility of developing preventative therapeutic approaches. et al. review published evidence evaluating the link between strenuous physical activity and the development of amyotrophic lateral sclerosis. The emerging consensus supports this environmental exposure as a risk factor for ALS and suggests that underlying genetic susceptibility factors are likely to be important.Chapman  The burden of ALS globally has increased substantially from 1990–2016, with a current estimated life-time risk of approximately 1 in 300.4Amyotrophic lateral sclerosis (ALS) is the most common subtype of motor neuron disease (MND), and the terms are often used interchangeably. It is a rapidly progressive and fatal disorder, with life expectancy commonly only 2–3 years from symptom onset.5–8 As such, ALS is an archetypal complex disease. Heritability studies suggest that ∼60% of ALS risk is genetically determined, and the remainder is environmental.6 Many of the genetic mutations linked to ALS, even those which are highly penetrant, are present for more than 50 years before disease onset. Indeed, the late age of onset of ALS points to a multistep process, in which genetic risk factors are penetrant only in the presence of additional environmental ‘hits’.9 The most common mutation linked to ALS is an intronic G4C2-repeat expansion of C9ORF72, which affects ∼10% of all ALS patients. The penetrance and severity of C9ORF72-ALS is markedly variable, which is consistent with interacting environmental factors.11 To date, the only confirmed epidemiological risk factors for ALS are male sex and increasing age, with onset most commonly between 60 and 75 years of age.13 Over the last decade, multiple reports have been released attempting to delineate the environmental risk factors predisposing to the development of ALS, however these have often been underpowered and have not led to significant advances in the field. The identification of a specific gene–environmental interaction could play an important role in disease prevention and a future personalized medicine approach. This review focuses on physical activity (PA) as a risk factor, and its interaction with the commonest known genetic risk factor, mutations in the C9orf72 gene, as a monogenic risk factor. There are currently no clear data on the link between polygenic genetic risk factors and PA as an exogenous environmental risk factor.The aetiology of ALS is considered to be due to a combination of risk-genotypes that interact with environmental factors over time, accelerating the neurodegenerative cascade.14 However, various studies have failed to confirm a link between PA and ALS.15–19 These conflicting conclusions may be explained by small study numbers, selection bias, no specific focus on risk-genotypes and the use of non-validated methods for quantification of historical levels of PA exposure.20 The controversy surrounding this hypothesis also relates to the benefits that regular exercise provides, for example for the cardiovascular system, leading to a potential survival bias.21 Most athletes and physically active individuals clearly do not develop ALS and it could be argued that the benefits of physical exercise outweigh the small potential risk of the development of ALS. However, a deeper understanding of this potential genetic–environmental interaction would allow for more personalized, focused advice for patients and family members and potentially could lead to the development of preventative strategies.The role of PA in the aetiology of ALS has been debated over several decades. ALS has been commonly documented in high profile athletes. This group are reported to have a higher incidence and lower age of onset of ALS, leading to the hypothesis that strenuous, repetitive exercise may represent an environmental risk factor.22 The present review aims to evaluate the recent evidence emerging since 2009, to provide a more conclusive answer as to whether or not PA is a factor in aetiology of ALS. An outline of each study’s main findings can be seen in Previous reviews focusing on PA and ALS concluded that there was insufficient evidence to make a definite conclusion on PA as a risk factor for ALS.36The papers for this review were identified by searching PubMed between 2009 and December 2021. The reference lists of these articles were searched to identify further relevant publications. The free text search terms were [(motor neuron disease) OR (MND) OR  OR (ALS)] AND [(exercise) or  or (PA) or (sport)]. The inclusion criteria included papers published since 2009 and in English language or that could be translated. The exclusion criteria included: papers focusing on paediatric patients; inability to access the full article text; papers which did not report clear outcomes; papers which could not be translated into the English language; papers discussing PA as a therapeutic measure rather than a risk factor; and systematic reviews. The final reference list was generated on the basis of significance to the topics covered in this review. The PRISMA flow diagram for this review is shown in 37 PA consists of either aerobic or anaerobic activity, with the involvement of specialized muscle fibres and motor neurons. Aerobic activity involves large muscle groups and can be maintained continuously. These muscles rely on oxygen to generate energy in the form of adenosine triphosphate (ATP) from amino acids, carbohydrates, and fatty acids. Aerobic PA or capacity relies on the capacity of the cardiorespiratory system to supply oxygen and the skeletal muscles capacity to utilize this oxygen. Aerobic capacity can be measured by peak oxygen consumption (VO2). Examples of aerobic PA are cycling, dancing, and long distance running and walking.38 Anaerobic PA involves high intensity, short duration exercise and uses the fast twitch muscles. Examples include sprinting or high-intensity interval training. The muscles experience a lack oxygen; therefore, the cells use glycolysis and fermentation to produce ATP. This produces less ATP than aerobic PA and causes an increase in lactic acid.38 In ALS it is this fast twitch (type IIb) anaerobic activity, i.e. vigorous activity, which is considered to be the predominant risk factor.38 Many studies have focused on different types of PA and their potential as an exogenous risk factor for ALS. However, the majority have not specifically divided the PA into aerobic and anaerobic activities, and this is likely to have a large impact and may account for some of the variability in conclusions reached in different reports.PA has previously been defined as activity that is ‘planned, structured and repetitive’ with an immediate or long-term goal to improve or maintain physical fitness.et al.31 conducted a case-control study focusing on the link between PA and ALS. They measured PA exposure during adult life using the HAPAQ. This questionnaire, developed in collaboration with the Medical Research Council Epidemiology Unit at Cambridge University, was validated in the separate Ely cohort of participants. HAPAQ was the first questionnaire instrument to measure historical levels of PA validated against repeated historical objective activity measurements.39 Harwood and colleagues31 used HAPAQ to quantify PA exposure in a regional cohort of 175 newly diagnosed ALS cases and 317 age and gender population matched controls. The main finding of this study was that participation in an extra 10 kJ/kg of daily PA  was consistently associated with an increased risk of ALS . Positive associations were reported for all domains of PA, with the strongest associations for individuals reporting higher levels of vigorous and exercise-related PA throughout adulthood.31 The data were collected retrospectively, increasing the risk of recall bias and over-estimation of activity levels, due to the social desirability of being more physically active. The nature and relative rarity of ALS make it difficult to avoid a relatively small cohort. Advantages of this study included blinding of the participants to the research hypothesis, therefore reducing recall bias. The careful selection of control participants and the use of a validated questionnaire were additional advantages.Harwood 40 Therefore, the use and accuracy of wearable devices needs to be studied further. In future studies, wearable devices could be used alongside the HAPAQ questionnaire to increase reliability and decrease bias.Despite being validated, there are still difficulties with a questionnaire-based approach to quantify individual levels of PA, due to personal bias. Another way of measuring PA could be via wearable devices. However, a recent review looked at the accuracy of wrist and arm-worn monitors in estimating energy expenditure and found that they differed depending on activity type.et al.,31 HAPAQ was conducted face-to-face, with life calendars of significant events to aid recall of historical levels of PA. This stringent approach increases the reliability of the study and allows us to draw the conclusion that PA is positively associated with the development of ALS, at least in this population of patients in the North of England.In the report of Harwood et al.27 set out to use Mendelian randomization (MR) as a means of overcoming limitations due to sample size. MR is a method whereby genetic variation within a population established at conception and which determines genetic liability for exposure to a risk factor of interest, serves as an instrument to study the effect of environmental modifiers on an outcome such as the risk of a specific disease, in this case ALS. It allows PA and ALS to be quantified in separate cohorts which removes the requirement for two measurements per patient, which can create a bottleneck for recruitment. In addition, MR does not suffer from problems with selection and recall bias that plague observational studies, although survival bias can still be a problem.41 Julian et al.27 discovered that high-intensity, frequent, leisure-time exercise increased an individual’s risk of developing ALS. Other forms of exercise, including occupational PA and movement measured by accelerometer, were not linked to the risk of ALS. The authors had predicted the result based on the observation that the most vulnerable motor neurons in ALS supply fast twitch muscle fibres.27 The specificity of this finding helps to counter the possibility that the result was confounded by survival bias.A disadvantage of the HAPAQ is that it cannot easily be applied to thousands of patients because it is relatively labour-intensive. Another recent study from Julian et al.27 also applied the HAPAQ in a smaller cohort of C9orf72-ALS patients (n = 17) where it was demonstrated that age of onset of C9-ALS was inversely correlated with levels of historical PA. This report did not find the same strong relationship in a larger group of sporadic ALS patients suggesting that there may be a specific interaction between the C9orf72 genotype and PA. These findings need to be validated in a larger cohort of C9orf72-ALS patients.Julian et al.15 assessed whether PA was implicated in the aetiology of ALS. The compulsory Swedish military service conscription physical exam, taken at the age of 18, was used for recruitment (n = 684 459). This increases the power of their study and reduces selection bias. The measurement of physical fitness used was the maximum load that conscripts could sustain for 6 min. This has good reliability and correlates well with other endurance tests.42 They concluded that weight-adjusted physical fitness  was a risk factor for ALS, but not PA per se. Despite the large sample size, PA was not assessed after the age of 18 years of age, hence the quantity or intensity of PA carried out in these individuals was not assessed longitudinally. The data were collected from men born between 1951 and 1965 and followed up until 31 December 2006, which would allow the oldest participant to be 55 years. Eighty-five males were identified as dying from ALS in this period. However, as ALS commonly affects individuals ∼60–75 years of age, a longer follow-up period would be needed to ascertain the true risk.13 The study design does not account for potential confounding variables, for example smoking or genetic factors. The authors do however highlight the possibility of an ‘athletic phenotype,’ implying that a particular genetic profile may contribute to sporting ability and the propensity to develop ALS.15A large-scale cohort study from Mattsson et al.24 focused specifically on vigorous PA in ALS participants. They used the National ALS registry, allowing for a large sample size, with 8739 participants completing at least one survey. They found a statistically significant difference between vigorous PA in the years 15–24 and 25–34 and diagnosis of earlier-onset ALS . They utilized a global PA questionnaire designed, validated, and evaluated by the World Health Organization. The lack of a validated questionnaire is commonly a limitation in many of the studies relating to ALS and exercise.A cross-sectional study conducted by Raymond et al.34 to assess whether ALS symptoms occurred in the dominant upper-limb or foot. In upper-limb onset patients they found a concordance for side of onset and dominant hand . However, in lower limb-onset patients this was not present (99/181 or 55% P = 0.234). They concluded that PA is linked to ALS, as physical demands on upper limbs are heavily influenced by limb dominance, whereas the commonest physical demand on lower limbs is standing, therefore this demand is distributed more equally between the right and left limbs. This study involved 502 participants and had a high response rate. The approach was prone to recall bias, as it is likely the symptoms would be noticed sooner in the dominant hand, due to greater use and awareness. The authors blinded the participants to the hypothesis, to reduce this potentially confounding element.A cohort study was conducted by Turner C9orf72 mutation.28 They compared C9-positive ALS patients (n = 143), C9-negative ALS patients (n = 1322) and healthy controls (n = 1322). Participants were assessed for 50 years pre-onset. The authors found that daily energy expenditure at symptom onset was significantly higher in the C9-positive  and C9-negative ALS groups  in comparison to healthy controls. The C9-positive group was a much smaller cohort, and therefore potentially this study was underpowered. This study highlights that, in this Dutch population, increased levels of physical exercise and having a C9orf72 mutation increased the likelihood of developing ALS. This paper only focused on the C9orf72 genetic subtype, and other genetic causes of ALS and polygenic risk factors were not considered.A recent prospective case-control study assessed the causality of lifestyle factors in the pre-symptomatic phase of ALS, in participants with the et al.27compared infrequent ‘strenuous sport’ or heavy DIY and frequent leisure-time PA. They found that infrequent ‘strenuous sport’ or heavy DIY were not significantly associated with ALS, but frequent leisure-time PA was linked.27 These forms of more static exercise would be most similar to activities such as resistance training. This is an area that requires further research.Many sports are not discussed in this review as they have not been researched extensively. It would be interesting to investigate whether resistance training such as used by body builders is a risk factor for ALS. Julian 35Overall, the evidence from the studies described above suggests that there is a positive correlation between total, strenuous PA over the life course and ALS risk, when a risk-genotype is present. Negative reported findings are likely due to underpowered studies, with unvalidated measures of PA, that have not focused on specific genotypes.43 There has been speculation about whether it is the type of PA undertaken by soccer players that increases the risk of ALS, or whether the association could be due to their increased risk of head injuries caused by heading the ball, the use of illicit performance-enhancing drugs or players continuous contact with grass .44–46Many professional athletes have developed ALS, with multiple reports specifically focusing on soccer players. Therefore, soccer specifically, rather than overall PA, has been suggested as a risk factor for the development of ALS. Soccer is predominantly an aerobic form of exercise, although anaerobic exercise occurs when players are performing sprints.et al.47 assessed non-genetic ALS risk factors, in 95 ALS cases and 135 controls. They found a slight increased risk of ALS associated with soccer at a competitive level , but an inverse association with sport in general . This implies that the specific type of exercise involved in competitive soccer carries a higher risk for the development of ALS. This study is limited as it does not explain how the exercise exposure data were collected and included only a small sample size of 95 ALS cases due to a low response rate (18.9%). The responses were also self-reported, allowing for recall bias. There was no information provided regarding position in the football team (which can be used to infer whether it was due to higher anaerobic or aerobic activity), risk of head injury or the time between playing soccer and diagnosis; this interval may not have been long enough.A recent case-control study conducted by Filippini et al.23 focused on NFL athletes, looking at incidence and mortality from ALS. They had large sample of 19 423 male former NFL athletes. They found that the incidence was four times as high as that of the general US population . Those diagnosed with ALS were also found to have a longer career (average of 7 years versus 4.5 years for those who did not develop ALS). Any other PA was not accounted for outside of NFL playing and the exercise itself was not quantified. Despite this, the large sample and positive findings imply that the association between NFL and ALS is most likely related to the large quantity of strenuous exercise performed over several years.A retrospective cohort study conducted by Daneshvar et al.14 assessed the risk of ALS in Italian soccer players (n = 7325), in comparison to road cyclists (n = 1701) and basketball players (n = 1973). There was a significantly increased risk of developing ALS at a younger age in soccer players, with a predilection for mid-field players. This risk was not seen in road cyclists or basketball players. Therefore, the type, frequency and mechanisms of PA appear important in relation to ALS risk. Basketball is mostly an anaerobic exercise, characterized by a lower maximum rate of oxygen consumption (VO2MAX), whereas road cyclists have a higher VO2MAX compared to soccer players. Previous studies have highlighted that midfielder soccer players require more aerobic activity and that they have a low anaerobic capacity.48 Therefore, it may specifically be intense aerobic activity that is a risk factor for ALS. This study does not explain how the amount of PA was quantified, or whether this was considered. Therefore, the soccer players may have had a higher frequency of strenuous exercise, over a longer time period. This study did not include genetic profiling, despite the C9orf72 genetic mutations having a relatively high prevalence in Italy compared to other countries.49 Another study focusing on soccer players, found that neurodegenerative disease was highest for defenders  and lowest for goalkeepers  implying that anaerobic activity is more important in the causation of ALS.26 This study had a much larger sample size of 30 704 males, therefore the results have higher statistical power. The authors also reported that the risk of ALS was higher, when soccer players have professional careers lasting over 15 years .26A prospective cohort study by Chio P < 0.0001).25 A case report described three amateur league soccer players from the same team in Southern England who all developed ALS.45 Another report focusing on mortality from neurodegenerative diseases of retired American football players, found that ALS was four times more common compared to the general US population.33 The specific cause of this link is not yet defined.Further reports strengthen this link between soccer and ALS. A recent prospective cohort study found that the mean age of onset of ALS in soccer players was 23.7 years younger compared to cases within the general European population  case-control study, assessing whether cross-country skiers had an increased risk of developing ALS in Sweden. Cross-country skiing involves a combination of sustained aerobic energy turnover and repeated work rates above the VO2peak (anaerobic activity). This differentiates cross-country skiing from other endurance sports.50 This type of PA burns the most calories per hour in comparison to other competitive sports.30 Both head trauma and use of illicit performance enhancing drugs are uncommon in skiing compared to soccer.30 This study was conducted to investigate whether it was soccer and head injuries specifically that increased the risk of ALS or whether it is the type of PA undertaken. Fang et al.30 found that faster skiers had a 4-fold increased risk of developing ALS . This supports the concept that strenuous PA is a risk factor for ALS in line with the work of Julian et al.27 previously mentioned. The analysis also adjusted for confounding variables, including education, employment, and region of residence. Additionally, the skiers who participated in more than four races  in comparison to one race had a higher risk of developing ALS.Fang 51 This offers an explanation for the increased risk of ALS described in the report from Fang and colleagues and strengthens the argument that strenuous, frequent PA enhances ALS risk, regardless of whether the specific type of PA predisposes to head injury. Moreover, the suggestion of a dose effect is good evidence which is consistent with the study described above linking the length of a soccer career to ALS risk.In preclinical studies moderate exercise has been shown to protect the neuromuscular system, whereas endurance exercise has been shown to promote oxidative stress and enhance motor neuron degeneration.52 Nuclear depletion and cytoplasmic aggregation of TDP-43 within motor neurons represent the hallmark pathology of ALS.53 However, it should be pointed out that the population of neurons affected by CTE is distinct from ALS. The evidence to date does not indicate that CTE is a cause for ALS, but rather that both conditions cause the development of TDP-43 proteinopathy.Several studies have assessed the relationship between the exposure to repetitive head injury, rather than PA itself, as a risk factor for ALS. Chronic traumatic encephalopathy (CTE) specifically is a form of neurodegeneration triggered by repetitive head injury. A 12-person case study of CTE showed that 10 of these cases had widespread TAR DNA-binding protein 43 (TDP-43) pathology, with three of these individuals developing progressive ALS several years before death.54–56 These symptoms could be related to CTE or ALS. Savica et al.16 conducted a cohort study focusing on whether the incidence of ALS was higher in high school American football players in the years 1946–56, before protective headgear was used. They found no increased risk of ALS in comparison to the control population. The severity of any concussion injuries was not detailed. This report also stated that the controls were ‘non-football’ playing, but it does not describe their PA levels. However, this study was focusing on head injury and not PA, and the report concludes that increased head injuries during football does not increase the risk of ALS.Multiple studies focusing on retired football players, found that players who had reported at least three episodes of concussion were significantly more likely to report cognitive symptoms in later life.et al.33 analysed the neurodegenerative causes of death among a cohort of professional American football players. They found a 4-fold increase in mortality from ALS in comparison to the general US population. They hypothesized that head injury was a risk factor and found that there was a higher mortality from neurodegenerative disease in those who had held speed positions compared to those with non-speed positions. They suggested that speed positions have a higher risk of concussion injuries, though this was not specifically evaluated. They did not document the numbers of head injuries or episodes of concussion. It should be noted that speed positions also require more strenuous, anaerobic activity which may relate to the increased risk of ALS.Lehman et al.27 used MR to link ALS to frequent strenuous PA. By definition, MR implicates a linear dose-response relationship, whereas the activities included in this exposure were diverse and will have had wide variance in their association with head injury. Therefore, head injury could not have caused this dose-response relationship, as it is very unlikely that this would occur as a linear function of PA.The study of Julian et al.47 conducted a case-control study in Italy. They found a positive association between ALS risk and head trauma . This study did not give methodological details of the instrument used to assess exposure to head trauma. It was also self-reported, allowing for recall bias. The sample size was small (n = 95) with a low response rate, which impacts upon the precision of the risk estimates. Another study focusing on American high-school football players, found that they had an increased risk of medically documented trauma, but not an increased risk of neurodegenerative disease.18 Therefore, trauma or head injury itself was considered unlikely to be a risk factor for ALS. The controls were other male athletes at high school, for example swimmers and basketball players. Therefore, this study does not address whether PA is a factor for the risk of developing ALS.Filippini To summarize, the evidence to date suggests that it is most likely the type of PA involved in the sports which can be associated with head injuries, that increases the risk of ALS, not the head injuries themselves. These studies highlight common problems with epidemiological studies in ALS. Conflicting results often arise because of low powered studies.51 When undergoing PA, the neuromuscular system experiences significant structural and functional alterations  and fibroblast growth factor (FGF) signalling pathways, which have been implicated in chronic adaptation to repeated exercise.61 The biological pathways implicated in this study are shown in et al.27).PA is a key factor in maintaining health across the lifespan. Regular moderate-intensity training reduces oxidative stress, decreases inflammatory markers such as interleukin-6, and helps to preserve cardiovascular fitness and brain function.erations . These rIn vivo studies in mice found that motor neurons respond to PA by synaptic reorganization, electrophysical changes and changes in neurotrophic signalling. These changes differed between acute and chronic exercise.62 Microarray technology and laser capture microdissection were combined to analyse response of motor neurons and skeletal muscle in 12 10-week-old (C57bl/6 strain) mice. Following a 6-week period of daily exercise mice were shown to upregulate 203 genes and downregulate 241 genes within motor neurons. The differentially expressed genes were mainly involved in signalling, the cytoskeleton, transcriptional regulation and synapse reorganization. The neuromuscular junction (NMJ) represents a specialized type of synapse. In mice following exercise a significant number of genes involving NMJ functioning, and development are transcriptionally altered.58 In chronic exercise for example, the catenin-cadherin system is activated. This is involved in cell-to-cell contact, neurite outgrowth and NMJ consolidation. Gene expression changes were also seen in the skeletal muscle following exercise. The gastrocnemius muscle showed upregulation of several protocadherins , which are involved in synapse formation. Therefore, chronic exercise alters synaptic reorganization and transcriptional regulation. Other important genes that are upregulated in muscle during exercise are Cntf and Lifr. These aid in the response to the physiological stress of intense exercise and mutations in these genes have been shown to be disease modifiers in ALS, leading to an increased risk and earlier age of onset.58It is difficult to study gene expression changes in response to PA in human muscle and motor neurons. 57 The authors highlighted that exercise induced a robust inflammatory response . PA also triggered early release into the circulation of pro- and anti-inflammatory proteins, increasing oxidative stress and propensity to apoptosis. They found that these gene expression changes were present acutely post-exercise, and the majority returned to baseline within 30 min post-exercise.57A recent report investigated the molecular choreography of acute exercise with longitudinal multi-omic profiling of plasma and PBMCs in 36 participants before and after controlled PA.in vitro astrocytes to cause motor neuron apoptosis, which has previously been implicated in the pathophysiology of ALS 48Drosophila model and (G4C2)48-MS2 rat spinal cord, G4C2 repeat expansions of C9ORF72 were shown to cause deficits in dendritic branching and therefore defective remodelling of the NMJ and synaptic dysfunction.66 This defective remodelling in patients with genetic mutations, such as those found in C9orf72, is likely to become a pathological phenotype when exposure occurs to the stress caused by exercise.58 In animal models of ALS, there is evidence of NMJ denervation and dysregulation of proteins involved in synaptic maintenance and plasticity.68 These studies highlight that, in the presence of genetic changes causative for ALS, the neuromuscular system may be unable to respond to the stress produced by PA in the same way as a healthy individual. This may explain the genetic–environmental interaction predisposing to the development of ALS.NMJ remodelling is an adaptive response to exercise. In previous studies in a pathological .71 These are used for anaerobic burst activity, high intensity exercise.71 Julian et al.27 in a recent MR study found that high-intensity, high-frequency, leisure-time exercise led to an increased risk of developing ALS. This type of activity involves the use of type IIb muscle fibres.62 Therefore, acute intense anaerobic exercise specifically stresses the motor neuron group most vulnerable to the disease process in ALS.Muscle fibres and motor neurons are sub-specialized for aerobic and anaerobic conditions. Skeletal muscle fibres are classified as either fast twitch  or slow twitch (type I) according to their contractile speed and metabolic properties.G93A mouse model of ALS showed an increase in mitochondrial mass and of mitochondrial DNA in different muscles during the course of the disease.72 Nicotinamide adenine dinucleotide dehydrogenase-tetrazolium reductase (NADH-TR) reflects the concentration of mitochondria within myocytes and succinate dehydrogenase (SDH) is a key enzyme in the Krebs cycle. The authors found that the activities of these enzymes in skeletal muscles were dramatically increased in the early pre-symptomatic stage of ALS (55 days).72 This hypermetabolism and its links to PA can also be seen in a case-control study evaluating the metabolic correlates of lifetime sport in ALS through brain 18F-FDG-PET scans.29 There was significant cerebellar hypermetabolism in the group who did not exercise compared to the group who did. This hypermetabolism correlated with more significant and widespread metabolic changes in areas known to be affected by ALS, the frontotemporal regions and corticospinal tracts. Despite this hypermetabolism, both groups expressed the same level of disability. The authors therefore concluded that participants who did not exercise coped better with the neurodegenerative process in ALS, inferring that PA has an additive effect to underlying changes in metabolism.A study in the SOD174 Morphological changes consistent with increased protein synthesis during exercise have been seen in the motor neurons of treadmill running rats.75 SDH was also increased in exercised skeletal muscle to compensate for the increased energy demand. If repetitive acute exercise is conducted , it may cause the motor neurons to be less excitable when at rest. Therefore, the effects continue when PA is stopped. These in vivo studies strengthen the argument that intense, strenuous PA combined with an underlying genetic mutation for ALS, can increase the risk of developing the disease.Professional athletes are known to be exposed to high levels of metabolic stress, and as described above, ALS appears to be more common within these individuals. Patients with ALS mutations have defects in energy metabolism, and an inability to respond to the additional metabolic stress of PA may be the cause of the propensity of these athletes to develop ALS at a younger age.in vivo study of SOD1G93A transgenic mice showed that running-based training increased their survival by delaying the onset of the disease. However, high endurance exercise had a detrimental effect on survival rate.77 These data suggest that high intensity exercise specifically may be detrimental in the presence of a SOD1 mutation—though this has not yet been explored in humans. Another study conducted in SOD1G93A transgenic mice showed a massive upregulation of energy generating pathways in motor neurons at the early pre-symptomatic stage,78 increasing our understanding of the impact that strenuous PA may have in an already stressed metabolic state.An 79 These findings may be due to the mutations affecting the slow-twitch muscles fibres primarily and therefore only being affected by specific sporting activities that trigger these specific fibre types. Overall, these studies highlight that specific high endurance PA and an ALS mutation, can potentially cause a toxic increase in energy generating pathways.Animal studies, for example in SOD1 mice exposed to exercise, have not consistently shown a detrimental effect of exercise on the disease phenotype. One study compared the effects of swimming-based training and moderate running-based training in the SOD1 ALS mouse model. They found that swimming significantly delayed motor neuron death, maintaining the fast phenotype in fast-twitch muscles and increased life span by ∼25 days.27 Abnormal biomarkers of oxidative injury within CNS tissues in ALS, have been shown in both post-mortem and in vivo studies.20 The elevated oxygen consumption and tissue metabolism required to perform vigorous exercise augments the generation of reactive oxygen species (ROS). Acute exercise is known to induce a transient increase in ROS, and individuals with chronic disease experience an exacerbation in oxidative stress following acute exercise in comparison to healthy individuals.32 During vigorous PA the normal adaptive processes seen in regular moderate exercise may not be achieved, causing deleterious effects of ROS.20 This excess of ROS can cause DNA and RNA modification, lipid peroxidation and oxidation and nitration of amino acids.80 This causes an accumulation of products of oxidative damage, causing further mitochondrial dysfunction and contributing to neurodegeneration , decreased ferric reducing ability (P < 0.001) and thiol groups (P < 0.001) in comparison to controls. This study indicates a decrease in antioxidant defences in ALS patients. The peroxisome proliferator-activated y co-activator 1α (PCG-1α) was specifically investigated, as it has previously been implicated in the pathogenesis of ALS.83 PGC-1α protects the mitochondria from oxidative stress by reducing the accumulation of ROS and apoptotic cell death.83 During inactivity PGC-1α is located in the cytoplasm, and it is activated during muscle contraction and relocates to the nucleus where it promotes the transcription of genes involved in skeletal muscle fibre remodelling, antioxidant defence, mitochondrial biogenesis and oxidative phosphorylation.32 The authors assessed different single nucleotide polymorphisms (SNPs) of PCG-1α. They found that lactate levels were significantly higher (P < 0.01 and P < 0.001) and greater protein oxidative products were found in patients with the AA (Ser482Ser) allele of PCG-1α compared to individuals with the GG (Gly482Gly) and GA (Gly482Ser) genotypes, which is consistent with greater exercise-related oxidative stress.32 In previous studies, the SNP change from G > A has been shown to inhibit the transcription of antioxidant genes and subsequently increase oxidative stress.84 Therefore specific mutations causing an increase in oxidative stress were found in ALS patients in comparison to controls.Pasquinelli 85 Overstimulation of glutamate receptors promotes an abnormal calcium ion influx into neurons, with adverse effects on mitochondrial function, increased oxidative stress and ultimately neuronal cell death.20 Motor neurons are particularly susceptible to excitotoxicity, given their high expression of calcium-permeable AMPA receptors lacking the GluR2 subunit and their relatively low expression of calcium buffering proteins.86 Overall, vigorous PA likely increases glutamate excitotoxicity and oxidative stress, hastening neurodegeneration in individuals with a genetic predisposition for the development of ALS.It has been proposed that vigorous PA might exacerbate glutamate excitotoxicity, which is known to contribute to motor neuron injury in ALS.5 At least 30 genes have been found to cause FALS, of which the most common is the intronic hexanucleotide expansion within the C9orf72 gene which is present in ∼40% of FALS cases and ∼7% of ALS cases with apparently sporadic disease. The penetrance of disease in the presence of C9orf72 mutations is not complete, although ∼99.5% of carriers will manifest disease by the age of 83 years.87 One study reported that 0.6% of healthy individuals in the UK carry an abnormal C9orf72 expansion.10 Most laboratories consider a GGGGCC repeat length of <30 in blood to be normal, with >30 repeats likely to be pathogenic.88 It is likely therefore that additional environmental factors may affect the penetrance of disease.90ALS is classified into sporadic (90–95%) and familial .et al.27 focused on enrichment of ALS genetic risk  with exercise-associated transcriptome changes. They highlighted the report of gene expression changes of known ALS genes post-exercise in PBMCs from normal individuals where C9orf72 was downregulated significantly following acute exercise .57 They also reported that, when using the validated HAPAQ questionnaire, the age of onset was inversely proportional to levels of historical PA for C9orf72-ALS  but not for non-C9orf72-ALS.Julian C9orf72 mutation.28 The authors found that daily energy intake at symptom onset was higher in the C9-positive  and C9-negative groups , concluding that a combination of increased PA and an underlying C9orf72 mutation can increase the risk of developing ALS. There were only 143 patients in the C9orf72 group, decreasing the power of the study. These studies only focused on one genetic cause of ALS and the interaction of PA with other genetic causes of ALS needs to be evaluated in the future.A case-control study assessed the effect of lifestyle factors on causation stratified by the 53 Two very recent studies have linked nuclear loss of TDP-43 to key splicing changes, leading to loss of function of genes including STMN2 and UNC13A, with consequences for clinical severity.92 TDP-43 is mislocalized to the cytoplasm, where it exhibits a toxic gain-of-function, contributing to neuronal injury and cell death.94 TDP-43 mislocalization is usually reversible in most model systems, however in both CTE and ALS, this mislocalization forms into potentially permanent pathological cytoplasmic aggregates.93 Importantly the quantification of these aggregates predicts the degree of neuronal loss.95 The specific effects that exercise has on TDP-43 aggregates is not currently known. The ability to specifically define the gene–environment interaction, involving exercise, that leads to this potentially irreversible TDP-43 mislocalization could allow a deeper understanding of the pathophysiology of ALS and aid with treatment discovery. One study previously attempted this; however, brain and spinal cord tissue was only obtained from 14 ALS patients.96TDP-43 is the hallmark pathology of ALS.The only epidemiological risk factors currently established for ALS are increasing age and male sex. The ability to establish further risk factors, would allow for more personalized medicine and potentially preventative strategies for this devastating disease.C9orf72 mutations; (iii) specific strenuous PAs that have been studied in the context of ALS include soccer, American football and long-distance skiing. Other sports with different anaerobic versus aerobic components should be studied in future; and (iv) the risk for ALS is not confined to impact sports with a high propensity for head injury.Key points emerging from this review include: (i) the balance of evidence over the past 13 years favours the conclusion that participation in frequent and strenuous leisure-time PA is a risk factor for the development of ALS; (ii) specific genetic backgrounds predisposing to exercise-induced ALS are being identified with early data highlighting the interaction with Current evidence for PA as a risk factor for ALS has increased in depth and quantity over the last few years. This review highlights the common problems faced in researching this topic, for example the relatively low prevalence of ALS, the challenges of measuring PA over the life-course, with unvalidated questionnaires and recall bias likely impacting the results obtained. The validated HAPAQ questionnaire has been used in a few of these studies and will allow historical PA levels to be measured with a reasonable degree of accuracy. Despite the issues the studies face, it is clear from the balance of all the evidence, that PA does play a part in the pathogenesis of ALS, when the participant has an underlying at-risk phenotype.C9orf72 gene. However, the majority of ALS has a polygenic architecture96 and future work will consider the interaction of exercise with the full spectrum of genetic changes linked to ALS (Genetic evidence to date on the role of PA as a risk factor for ALS, has focused on the most common genetic cause of ALS—mutations of the d to ALS . The advIt is likely that specific forms of exercise are important, for example anaerobic reactions in strenuous activity, and factors such as sports-related head injury are not. In the future, large scale studies focusing on specific at-risk genotypes and PA should be conducted to confirm this association . This wiawac470_Supplementary_DataClick here for additional data file.


Wolbachia is one of the most common bacterial endosymbionts, which is frequently found in numerous arthropods and nematode taxa. Wolbachia infections can have a strong influence on the evolutionary dynamics of their hosts since these bacteria are reproductive manipulators that affect the fitness and life history of their host species for their own benefit. Host–symbiont interactions with Wolbachia are perhaps best studied in the model organism Drosophila melanogaster, which is naturally infected with at least 5 different variants among which wMel and wMelCS are the most frequent ones. Comparisons of infection types between natural flies and long-term lab stocks have previously indicated that wMelCS represents the ancestral type, which was only very recently replaced by the nowadays dominant wMel in most natural populations. In this study, we took advantage of recently sequenced museum specimens of D. melanogaster that have been collected 90 to 200 yr ago in Northern Europe to test this hypothesis. Our comparison to contemporary Wolbachia samples provides compelling support for the replacement hypothesis. Our analyses show that sequencing data from historic museum specimens and their bycatch are an emerging and unprecedented resource to address fundamental questions about evolutionary dynamics in host–symbiont interactions. However, we also identified contamination with DNA from crickets that resulted in co-contamination with cricket-specific Wolbachia in several samples. These results underpin the need for rigorous quality assessments of museomic data sets to account for contamination as a source of error that may strongly influence biological interpretations if it remains undetected. Wolbachia pipientis  haplotypes are nonrandomly associated with specific Wolbachia types  sequencing to newly sequence genomic DNA of 6 freshly collected isofemale lines from wild populations in Portugal and Finland that were naturally infected with either the wMel or the wMelCS Wolbachia types and 3 lab stocks that had been artificially infected with wMel, wMelCS, and wMelPOP  or previously tested positive for infections with wMel (n = 20) or wMelCS (n = 2)  and wMel (wYak Wolbachia type (NZ_VCEF01000001.1) as an outgroup for phylogenetic tree reconstructions.In total, 69 genomic data sets were included in the present study  online: pecimens  were teslPOP see  online.  (n = 2) , for phy (n = 2)  online. ata sets , 2016 anata sets , which rg wMelCS  that werg wMelCS  for phyltype see  online. and wMel , we usedwMel, wMelCS, or wMelPOP by means of ONT technology, we confirmed their infection status and infection type with PCR using Wolbachia-type-specific VNTR-141 primers and PCR conditions as described in Prior to whole-genome sequencing of 9 contemporary isofemale lines  online iD. melanogaster mt genome (NC_024511.2) and only retained reads that matched the references in the database. Then, we blasted 100,000 randomly drawn filtered mt-specific reads against a local copy of the NCBI nt database using the blastn algorithm . Based on the target sequence ID (sseqid) and the NCBI TaxID (staxids), we obtained information on the best hits at the genus level and further analyzed the results in R , to enrich the raw sequencing data of the 5 samples and the newly downloaded data for reads specific to supergroup B Wolbachia. Finally, we mapped the filtered paired-end reads of each data set against wMeg using bwa mem , we calculated average read depths (RDs) for all Drosophila chromosomes and symbiont genomes. Using this information, we estimated relative Wolbachia titers for a given sample by dividing the average RD at the Wolbachia genome by the average RD across all Drosophila autosomes  infected with Wolbachia , (ii) uninfected , or (iii) with unclear status . These thresholds are necessarily arbitrary, albeit informed by a strong correlation between RD and coverage in the historic samples , wMelCS (RefSeq: NZ_JACSNK000000000.1), and wMelPOP (RefSeq: NZ_AQQE00000000.1) and only retained reads that matched the references in the database. For the Illumina sequencing data of historic and contemporary samples downloaded from NCBI SRA, we mapped paired-end reads using bwa mem . Conversely, we used minimap2 . We only considered a position diagnostic whenever the RD was ≥2 and the allelic state was unambiguously identified based on a posterior probability as explained above. Two of the samples with uncertain infection status (19SL2 and 18SL6) did not have coverage at any of the diagnostic markers and thus had to be excluded.When comparing 33 contemporary infected samples with known ectively  online. Wolbachia-specific reads with Kraken using a custom database consisting of wMel, wMelCS, and wMelPOP reference genomes as described above for de novo assembly with SPAdes  as a backbone to align and orient the raw contigs with nucmer of the MUMmer package , we then combined these contigs into a single scaffold and filled the gaps between each pair of consecutive contigs with a string of 10 N's. Moreover, given that the bacterial genome is circular, we anchored the newly assembled scaffolds at the start point of the reference genome and shifted protruding sequences to the end of the scaffolds.Finally, we used the published  v.3.23; . Based oWolbachia based on phylogenetic inference:We employed 2 complementary approaches to explore the evolutionary history of R , we converted the VCF file generated in chapter 2.4 to the PHYLIP format, only considering biallelic polymorphic positions where the posterior probability of the most likely genotype was >50 and of the alternative genotype was <30. In addition, we only included positions with more than 5-fold RD in each of the samples and removed samples, with more than 50% missing information across all SNPs. We used a total of 279 aligned SNP data to reconstruct a ML tree based on the GTR-CAT substitution model from 20 randomly drawn MP starting trees using RaXML . To this end, we included all historic samples irrespective of their infection status. We then performed a phylogenetic analysis with this data set using RaXML as explained above. history . This alWolbachia and mt SNP data sets that included the same samples and performed a phylogenetic analysis as described above. Then, we used the function as.dendrogram from the R package phylogram  test as implemented in the R package ape  and 3% (19SL23) of the total reads for a given sample were similar to Wolbachia of supergroup B. To test if these reads indeed originate from Gryllus-specific Wolbachia rather than from the Drosophila host, we further employed a phylogenetic analysis based on fragments of the Wolbachia-specific wsp locus. Besides two of the historic samples 19SL3 and 19SL22, which had sufficient coverage (>10%) at the wsp locus, we further included 1 Wolbachia sequence of G. campestris from the NCBI GenBank . Wolbachia titers relative to the Drosophila host in museum samples varied dramatically and ranged from 0.001:1 up to 5:1 . Conversely, we considered 7 historic samples as uninfected. Besides the 2 aforementioned infection types, we identified 8 samples with uncertain infection status, which were characterized by low RD (2- to 9-fold), which covered only parts (15% to 50%) of the reference genome and which had quite low relative titers (0.02:1 to 0.2:1).Based on these results, we qualitatively classified 10 historic samples as unambiguously infected with Wolbachia genome. Although we find similarly low or even lower relative titers in uninfected contemporary samples  which may be characterized by different sequencing errors with respect to GC content see . This coGC: 43%;  and Wolbnditions .Wolbachia variants identified in the historic samples are of wMel and/or of wMelCS type. To this end, we took advantage of our genomic data set from recent samples with known Wolbachia type and identified 67 diagnostic marker SNPs that were fixed for different allelic states in the 2 types. We were able to investigate 11 historic samples that did not contain foreign arthropod DNA contamination, where at least 3 SNPs contained allelic information due to sufficient RD. All samples of which 10 were previously characterized as infected and 1 was of uncertain infection status based on relative titer were unambiguously identified as infected with wMelCS , which further emphasizes tight links between the evolutionary histories of mitochondria and Wolbachia. The only major difference between the 2 trees is the placement of sample 18GP9, which is located at the base of the wMelCS cluster in the Wolbachia tree but located within the wMelCS cluster in the mt tree. Given that the discordance is occurring only on branches with low bootstrap support in either of the 2 trees, we assume that these few inconsistencies are not the result of horizontal transfer but rather of low resolution at the sequence level.Previous analyses have unveiled a close link between the evolutionary histories of  species . Both thchondria . This paWolbachia type; i.e. historic and contemporary wMelCS-infected samples formed a monophyletic cluster that was distinct from the contemporary wMel samples, which were subdivided into 4 monophyletic clusters that correspond to the wMel groups previously defined in wMelCS cluster. This provides further evidence that modern mt haplotypes that are nowadays associated with wMel were absent or rather rare in Northern Europe, or at least in the geographic area covered by our historic data set, 100 to 200 yr ago. Consistent with this hypothesis, we only found 1 sample (18DZ5) among the 25 historic samples, which was of a wMel-specific mt haplotype. Our analyses of mt haplotype variation in historic samples thus further support a recent sweep of wMel in worldwide populations. However, more data from other historic samples in Europe and North America are needed to narrow down the putative origin of the wMel invasion and to better understand the expansion route of the sweep that led to a turnover not only in Wolbachia but also in the associated mt haplotypes.Consistent with our expectations and with previous data , we founWolbachia-specific contigs were within the range of the expected total genome size (i.e. approximately 1.26 Mb). In contrast, cumulative contig lengths for the other samples were markedly shorter and ranged from 26 to 590 kb in spite of RD > 10-fold. This suggests elevated levels of sequence degradation in these samples, which is further indicated by N50 values < 100 bp and GC ratios, which are markedly higher than the wMel-specific GC ratio of 35.2%.Finally, we further used the sequencing data of the 10 historic samples, which we classified as infected, and of the 9 newly sequenced contemporary samples for de novo genome assembly. Assembly quality, which we assessed based on the BUSCO approach and on general assembly statistics, such as total number of contigs, cumulative contig lengths, length of the largest contig, N50 and N90, varied highly among the different data sets  online. Consistent with the variable data quality of the investigated historic samples, we found large differences in the proportion of complete BUSCO sequences for the different assemblies . The 5 hwYak reference genome as an outgroup and reconstructed a ML tree based on the concatenated gene alignments, which yielded a total length of 178,150 bp. The resulting tree . Such new data sets will enable studies to comprehensively quantify and investigate evolutionary change by comparing historic to contemporary genomic data in model and nonmodel organisms preserved in museum collections. However, given the low amounts of DNA and the high levels of degradation, museomic data sets are also particularly prone to contamination and thus require rigorous and novel approaches to test for data quality. This is illustrated by contaminations with human and cricket DNA resulting in co-contaminations with cricket-specific Wolbachia that we detected in several samples of the historic data set. Our findings demonstrate the need for thorough quality assessments to avoid drawing false biological conclusions.In summary, we, for the first time, identified and characterized historic axonomic  and geogaxonomic , our anamsad258_Supplementary_DataClick here for additional data file.


This article was migrated. The article was marked as recommended.First-year medical students are at risk for burnout despite resources available to help them manage stress. In 2015, a structured peer-mentorship program was created at our institution for incoming medical students (M1s) by second-year medical students (M2s) with a goal of reducing risk factors for burnout; a secondary goal was to improve the mentoring skills of M2s. Over the course of the year, we surveyed M1s about their anxiety, prioritization skills, and work-life balance; M2s from the previous year’s unstructured peer-mentorship program served as a control group. 164 M1s and 164 M2s participated in this program. Among M1s, a structured peer-mentorship program significantly reduced anxiety levels (p=<0.01), improved prioritization skills (p=<0.01), and facilitated greater awareness of the importance of striving to maintain work-life balance (p=<0.01). M2s felt neutral-to-agreeable in their ability to provide guidance, refer students for help, and remain invested in their mentees. A structured peer-mentorship program, therefore, may reduce anxiety, improve prioritization skills, and emphasize the importance of work-life balance among M1s, elements that have been associated with reduced rates of burnout. Furthermore, this program can augment the professional development of M2s by motivating them to maintain longitudinal mentoring relationships with underclassmen. The medical school environment poses unique challenges to students regarding work-life balance, career exploration, and acquiring clinical skills and knowledge . These cA study of nearly 4,300 students at seven medical schools found a 50% burnout rate, suggesting that many students could benefit from additional support .Burnout can be attributed to several factors, including increased anxiety, poor prioritization skills, and work-life imbalance . AnecdotOur institution had a longstanding peer-mentoring program  designed to facilitate interactions between second-year medical students (M2s or “big sibs”) and first-year medical students (M1s or “little sibs”). This program was unstructured, denoting no formal requirement for sibs to meet or direct responsibility for the big sib to mentor his or her little sib. Feedback about the program was mixed. While some of the sib pairings resulted in longitudinal mentoring relationships, many sib relationships did not persist beyond the first month of school. This observation could be attributed in part to increased academic demands from M2s and reluctance of M1s to seek advice from their big sib.In 2015, we implemented a structured peer-mentorship program designed to promote greater interaction between sibs throughout the academic year and enhance mentoring. Our goal was to improve the transition to medical school for M1s by reducing burnout risk as assessed through their anxiety, prioritization skills, and importance for maintaining a work-life balance. A secondary goal was to improve the mentoring skills of M2s.Program DesignOur peer mentorship program was structured as follows: incoming M1 students were randomly assigned into one of four societies by the medical school administration. A society represents a community of students across all years of training intended to facilitate interclass mentoring sessions and social events. Student leaders in the societies paired each M1 with an M2 based on similar undergraduate institutions and degree backgrounds. Student participation was required unless exempted by their society’s faculty mentor. Throughout the academic year, M2s received emails every four to six weeks containing talking points that prompted them to interact with their little sib as shown inEach module was paired with a talking point focused on predictable challenges and career guidance between siblings. These talking points were included in the email prompts received by the M2s to discuss with their little sib. These talking points were formulated by an ad hoc committee of students and faculty focused on addressing times of predictable stress in the M1 academic year. Students were encouraged to interact in person. Other alternative modes of acceptable communication between siblings included texting, calling, emailing, FaceTiming, and Skyping.This student-directed initiative was designed to encourage M2s to remain engaged with their little sib and provide a greater number of M1s with the opportunity to have a meaningful mentoring relationship with an upperclassman. Of note, M2s did not receive any mentoring training prior to the initiation of the structured peer-mentorship program.Study Design and Data AnalysisThis was a prospective longitudinal study of M1s and M2s at Northwestern University Feinberg School of Medicine (FSM) in Chicago, IL, USA. Incoming M1s were the experimental group, participating in the new structured peer-mentorship program; M2s, having participated in an unstructured peer-mentorship program as M1s the year before, served as the control group.The study population included all M1s and M2s enrolled at FSM with a big or little sib, respectively. Students were not included if they did not participate in the peer-mentorship program. Approval of this study was exempted by the Northwestern University Institutional Review Board.During the academic year, M1s and M2s were evaluated with voluntary, anonymous Google surveys using a 5-point Likert scale  according to the timeline provided inEach letter in the timeline in The M1 year was split into two blocks in order to achieve a midpoint and final assessment of the program by M1s. During these program assessments, we surveyed M1s on their number of mentoring discussions between siblings, their reasoning for fewer than 3 mentoring discussions between siblings per block, and their descriptions of any conflicts between siblings (survey E). A mentoring discussion was defined as an in-person meeting between siblings. If sibling pairs were unable to meet in person, other communication methods were used but were not surveyed. Two or fewer mentoring discussions per block represented less than optimal participation in the structured peer-mentorship program.Prior to the initiation of the new structured peer-mentorship program, we surveyed M2s about their experience as M1s with the unstructured peer-mentoring program, focusing on anxiety, prioritization skills, and work-life balance (survey B). After one year in the structured program, we surveyed M2s on their mentoring skills by assessing their comfort in giving advice and guidance, motivation to help their sib, capabilities with referring sibs to additional resources, and identification of areas for growth in their little sib.At the beginning of the academic year, we assessed M1s to measure their anxiety and comfort level after being assigned to a big sib (survey A). At the end of the year, we surveyed them about their anxiety, prioritization skills, and importance of work-life balance (survey C).p < 0.05 was determined to be statistically significant. The total number of responses were recorded with the mean, median, and standard deviation calculated for each question asked in all surveys.Data from survey B was compared with the results from survey C using an unpaired student’s t-test whereA total of 164 M1s and 164 M2s participated in the structured peer-mentorship program. Only one M2 was excluded from the sibling program.The duration of the unstructured program was approximately 10 months (August 2014 - May 2015) compared to the structured program that lasted for 7 months (August 2015 - February 2016). The structured peer-mentorship program ended earlier than anticipated from the study design due to protected time included in the M2 curriculum for dedicated USMLE STEP 1 studying. Consequently, M2s did not receive email prompts to contact their little sib during the last three months of the M1 academic year. The response rates for each survey administered can be found inM1s responded to a two-question survey prior to arrival on campus for matriculation. This survey focused on reduction of anxiety and comfort level seeking advice related to transitioning to medical school. This data is shown inM1 responses were recorded based upon student agreement with (A) ease of anxiety and (B) development of comfort level in seeking advice from their assigned big sib. At the end of block 1, M1s responded to survey E, with over 33% (n = 37) of respondents reporting 4 or more mentoring discussions. The majority of M1s with less than 3 mentoring discussions responded that informal communication had replaced formal meetings or more than one mentoring session per module was unnecessary. Only one M1 reported significant conflict with their big sib. In block 2, a decreased number of M1s responded to survey E compared to block 1. A portion of M1s stated their big sib or themselves performed less outreach as one reason for less than 3 mentoring discussions. No sibling conflicts were reported during block 2. These results are presented inp = 0.0004, 0.001, and 0.004, respectively, in favor of M1s utilizing a structured peer-mentorship program.First-year medical students at our institution appear to have benefited from having a structured peer-mentoring program. Our data suggest that, compared to an unstructured peer-mentorship program, introducing guidance via talking points and prompting regular mentoring sessions significantly reduced anxiety, improved prioritization skills, and raised awareness of the importance of striving to maintain work-life balance-factors associated with a risk of burnout. To our knowledge, this study represents the first assessment of a structured peer-mentoring program aimed at reducing the burnout components in first-year medical students.These benefits may be attributed, in part, to a program that prompted M2s to reach out to M1s at times of predictable stress during the academic year, such as preparing for their first objective structured clinical examination (OSCE) or written examination. Over the academic year, students reported having multiple mentoring discussions between siblings, especially early in the year. The reduction in mentoring discussions later in the year may suggest that students had adjusted to school and anxiety levels had declined from the start of medical school. Additionally, the observed decline in discussions could further be explained by the absence of the M2s for their protected time to prepare for USMLE STEP 1. However, evidence of unprompted discussions between siblings was noted inOur results indicate that having a big sib reduced the anxiety of M1s. Interestingly, this benefit was notedeven beforestudents arrived at school or had met with their big sib. Additionally, having a big sib was associated with a willingness of M1s to seek advice about challenges related to transitioning to medical school. These observations emphasize the high levels of anxiety among incoming students and offer a readily available solution to alleviate it.A surprising finding was that, throughout the academic year, only one M1 reported a poor relationship with his or her big sib. The outcome of this conflict remains unknown from our data. There may have been other sub-optimal sibling pairings that were not captured in our data due to a response rate of 55% and 48% for blocks 1 and 2, respectively. It is also possible that student concern over exposing conflicts between siblings for review by their peers and faculty may have inhibited additional reporting of poor relationships.Finally, M2s also appeared to benefit from this program. Our data suggest that participating M2s were motivated to assist their mentees. For many M2s, this role may have been the first time they have functioned as mentors. This experience appears to have encouraged them to model constructive behavior, which may improve their performance later in the apprenticeship model of training (Stalmejjer, 2015;There are several limitations to this study. First, data obtained was from a single institution, limiting the generalizability of our findings. Our data is further limited by the use of non-validated surveys in assessing self-reported anxiety, prioritization skills, and work-life balance understanding in contrast to objective measurements of these variables. Second, our data is limited to a single, truncated year. While our findings were promising, additional data is needed to further support our findings and its reproducibility. As part of this study, a declining response rate may represent selection bias leading to a non-representative sample of M1s and M2s. Third, we surveyed M2s about their experiences as little sibs at the start of their second year of medical school. This delay in surveying M2s after completing their first year of medical school introduces the potential for recall bias in the control group.As this program evolves, we anticipate M1s who participated in a structured program may perform better as M2 mentors. These M2s may feel more confident when helping their little sibs address common challenges of the first year in medical school compared to M2s who had participated in an unstructured program. Subsequent iterations of the Sibs Program may also show even greater reductions in anxiety levels and improvements in prioritization skills and work-life balance. In addition, it would be helpful to collect data regarding the barriers to mentoring identified by M2s that could be used to develop strategies to improve their mentoring skills.Compared to an unstructured peer-mentoring program, a structured peer-mentoring program was associated with self-reported reductions in anxiety, improved prioritization skills, and greater awareness of the importance of striving for work-life balance among first-year medical students. These elements can ease the transition into medical school and are associated with reduced risk of burnout. Incoming medical students have positive associations with being assigned a peer mentor, and second-year medical students report that serving as a peer mentor motivated them to help mentees.•First-year medical students are at risk for burnout despite commonly available resources to help them manage stress.•Compared to an unstructured peer-mentoring program between first- and second-year medical students, a structured program was associated with self-reported reductions in anxiety, improved prioritization skills, and greater awareness of the importance of striving for work-life balance among first-year medical students.•Second-year medical students reported that serving as a peer-mentor motivated them to help their mentees and facilitated a sustained longitudinal mentorship.Gabriel A. Arenas is an alumnus of the Northwestern University Feinberg School of Medicine, where he earned his medical doctorate in 2018. He is currently an obstetrics and gynecology resident at the University of Chicago Medical Center interested in medical education and mentorship.Gregory E. Brisson is a clinical assistant professor of internal medicine at Northwestern Memorial Hospital. He is also the leader of the academic societies within the Northwestern University Feinberg School of Medicine. This review has been migrated. The reviewer awarded 4 stars out of 5 Thanks for the opportunity to review this article. This is an educational intervention study compared the two groups who underwent un structured and structured peer mentorship program and ti was concluded that the first-year medical students appear to have benefited from having a structured peer-mentoring program which is evident statistically by unpaired t test between M1 and M2 students mean values. Two queries / suggestions to the authors1. Was there any interim analysis conducted or any pre and post comparison for M1 students done in this study?2. Complete Study protocol can be added as a supplementary file to get more clear picture on – the study population, recruitment, follow up, outcome surveys and analysis. Thanks regards Dr. A. BalajiReviewer Expertise:NANo decision status is available This review has been migrated. The reviewer awarded 4 stars out of 5 I enjoyed reading this paper about the big sib mentoring program at the Northwestern University medical school and agree with the authors that peer mentoring is an effective approach to enhancing wellbeing among junior medical students.The abstract summarizes the program and results well.The introduction provides a good lead in to why the program was designed and the need for this study. The problem is stated, the gap in literature mentioned and the goals of the program are reasonably clear.Under methods, the program has been described well. I really like the approach where the students are placed into societies and the sibling program constructed within the society. This provides many opportunities for engagement and society faculty availability for guidance. The talking points are also well done to ensure that the siblings have a framework for their conversations.Tables 1 and 2 provide informative details about the structure of the program and evaluation time points.The results section only consists of graphs and tables without a concise summary of what the most important results were. A lot of data have been collected and I am not entirely sure all of them are necessary to inform readers about the impact of the program. I could have been convinced of its effectiveness with less data. I would have actually liked to have seen some narrative or open-ended comments which would have been really meaningful.Overall the paper is well written, well referenced and would be of interest to all educators designing peer mentoring programs for undergraduate or postgraduate trainees.Reviewer Expertise:NANo decision status is available