Brain-gut-microbiota interactions in sleep disorders

Depression disorder and anxiety disorder

Numerous studies indicate that the comorbidity of sleep disturbances with neuropsychiatric diseases has unique microbiological properties in patients (5, 53, 54). The role of gut microbiota as a mechanistically involved factor in comorbidity warrants further exploration. Prior research has demonstrated a causal relationship between some microorganisms (Roseburia, Veillonellaceae, etc.) and depression (5). Zhang et al. discovered that the makeup of gut microbiota correlates with sleep disturbances in major depressive disorder (MDD), with four genera associated with sleep quality and Intestinibacter linked to sleep severity (53). The composition of specific gut bacteria, including Flavonifractor and Lachnoclostridium, showed a strong association with daytime functional performance (54). Prolonged sleep deprivation (SD) can result in both depression and alterations in gut microbiota (55). α-diversity is diminished in individuals with depression and anxiety accompanied by insomnia, while Bacteroides exhibited a positive connection with Pittsburgh Sleep Quality Index (PSQI) levels in the noninsomnia cohort (56). SD induces anxiety-like behaviors in rats, along with alterations in the gut flora (57). These results suggest that alterations in gut microbiota may serve as an indicator for comorbid sleeplessness associated with depression and anxiety disorders.

Potential mechanisms underlying the bidirectional relationship between sleep and gut microbiota

The microbiota-gut-brain axis coordinates the sleep process in humans and rodents through several bidirectional communication routes, including metabolic, neurological, and immunological pathways (Figure 3). The vagus nerve detects and relays signals related to metabolic and immune functions through molecules such as hormones, fatty acids, and cytokines. Furthermore, metabolites and other bioactive substances from the gut can pass through the intestinal and blood-brain barriers, providing an additional mechanism for gut-brain communication. This section explores the potential pathways through which the microbiota-gut-brain axis influences sleep and the gut microbiome.

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Bile acids

BAs are cholesterol-derived metabolites essential for solubilizing dietary lipids and fat-soluble vitamins in the small intestine, facilitating their absorption or excretion. Synthesized in the liver as primary bile acids and stored in the gallbladder, they are released into the gut, where bacterial metabolism transforms them into secondary bile acids. These secondary bile acids act as signaling molecules, regulating metabolic and immune processes while shaping gut microbiota composition.

Circadian disruption has been shown to affect BA metabolism by altering BA homeostasis and enterohepatic circulation (63). Studies indicate that circadian disturbances influence BA metabolism, with sleep disruption significantly increasing the BA pool size—comprising bile acids in the gallbladder, intestine, and liver—particularly observed at ZT2 in sleep-disturbed mice (64). These findings highlight a strong link between sleep and BA regulation, suggesting potential impacts on metabolic processes. A prebiotic diet has been shown to modulate bile acid metabolism by reducing the levels of certain secondary and conjugated bile acids, while increasing cholic/taurocholic acid in feces. Prebiotic intake represents a promising strategy for enhancing gut health and mitigating the effects of circadian disturbances on sleep (65). Chronic insomnia is associated with alterations in gut microbiota composition and specific bile acid profiles, including elevated levels of MCA and NorCA and reduced levels of IsoLCA, LCA, and UDCA (8). These findings suggest that the microbiota-bile acid axis plays a critical role in the impact of chronic insomnia on cardiometabolic health, offering a potential target for therapeutic intervention.

Short-chain fatty acid

SCFAs, including butyrate, acetate, and propionate, are metabolites produced primarily by gut microbiota fermentation of dietary fibers. Sodium butyrate has demonstrated efficacy in enhancing sleep quality among patients with active ulcerative colitis, as evidenced by a double-blind randomized controlled trial, underscoring its dual function in promoting gut health and improving sleep (66). In animal studies, butyrate supplementation alleviated the inflammatory response and memory impairment induced by SD, pointing to its protective effects on cognitive and physiological health (67). Furthermore, oral melatonin supplementation, by modulating gut microbiota and its propionic acid metabolite, improved skin damage linked to circadian rhythm disruptions (68). Butyrate supplementation improved sleep restriction–induced obesity in mice (69). These studies suggest that butyrate has extensive ameliorative effects on various abnormalities caused by sleep disorders. However, the relationship between SCFAs and sleep efficiency appears complex. Enhanced physical activity lowers SCFA levels in older individuals suffering from insomnia. Older adults with insomnia demonstrated a negative association between sleep efficiency and fecal SCFA concentrations, including acetate and butyrate, possibly due to altered SCFA metabolism with aging (70). Sleep time has been positively correlated with fecal concentrations of total SCFAs, including acetate and propionate (71). Additionally, children with lower wake times after sleep onset exhibit higher fecal propionate levels. This suggests that SCFAs may influence sleep continuity (72). These studies suggest that SCFAs are associated with sleep across different age groups. A deficit in short-chain acyl-coenzyme A dehydrogenase (encoded by acids) in mice resulted in a distinct reduction in theta frequency during paradoxical (REM) sleep, thus indicating a role for SCFAs in the modulation of brain activity during sleep (73). Sleep fragmentation can indeed influence the levels of SCFAs. Increased acetate levels have been observed in patients with OSA, especially when coexisting with type 2 diabetes, a chronic sleep fragmentation mouse model exhibited elevated acetate levels in hypothalamic astrocytes as a protective mechanism (74). These findings suggest that SCFAs like acetate may mitigate metabolic and cognitive impairments associated with disrupted sleep.

Gamma-aminobutyric acid

Several GI bacteria, including strains from Lactobacillus and Bifidobacterium, possess the gene encoding glutamate decarboxylase, which facilitates the production of gamma-aminobutyric acid (GABA) (75, 76). These bacteria can elevate GABA levels in the enteric nervous system, highlighting their potential significance in gut-brain communication. Vancomycin treatment in mice led to a significant decrease in cecal GABA levels (77), suggesting that antibiotic disruption of gut microbiota composition directly affects GABA production and its availability in the GI tract. Studies using electroencephalography (EEG) have shown that oral GABA intake induces changes in brain responses compared to control substances such as water, L-theanine, or a dextrin placebo. This indicates that GABA produced or supplemented via the gut may influence central nervous system activity (78). Further research is essential to clarify how gut microbiota and GABA-homocarnosine metabolism play a role in sleep behaviors.

Serotonin and tryptophan

Over 90% of the body's 5-HT is synthesized in the gut, with gut bacteria serving as major producers, especially in the neonatal intestine (79, 80). Serotonin concentrations peak during awake, diminish throughout slow-wave sleep, and reach their nadir during REM sleep across several brain areas, including the hippocampus and cortex (81). This rhythmic fluctuation highlights serotonin's regulatory role in the sleep-wake cycle. Tryptophan, the unique precursor for serotonin and melatonin biosynthesis, is crucial for sleep regulation.

Microbiota-depleted mice have elevated tryptophan levels but reduced serotonin, indicating that gut microbiota are essential for converting tryptophan into serotonin (13). Stress-induced changes in tryptophan metabolites are microbiome-dependent and localized to the gut, further linking gut health to serotonin production (82). Microbial metabolites of tryptophan, such as ligands binding to receptors like AhR (83, 84), TRPA1 (85), and PXR (86), modulate gut barrier integrity and host tryptophan metabolism. These interactions underscore the microbiota's role in maintaining gut and systemic health. Tryptophan is the unique precursor for the biosynthesis of serotonin and melatonin, which are crucial regulators in the sleep-wake cycle. 5-hydroxytryptophan supplementation can improve certain sleep quality components and the gut microbiota composition, particularly in older adults with poor sleep (87). This suggests a bidirectional relationship between gut health and sleep quality.

Melatonin

The GI tract is the most significant extrapineal source of melatonin, with concentrations reaching up to 400 times those found in plasma (88). SD reduces melatonin levels in both the gut and plasma, altering the composition of the gut microbiota (89, 90). In sleep-deprived mice, lower plasma melatonin levels were associated with decreased colonic Card9 expression, leading to colitis and gut microbiota dysbiosis (91). Exogenous melatonin also ameliorated neuropsychiatric symptoms caused by chronic SD (92), highlighting its potential for treating sleep-related mental health issues. Melatonin restores circadian rhythm homeostasis in the gut microbiota disrupted by sleep restriction. This suggests a protective role against the systemic effects of poor sleep (93). These findings emphasize the critical role of melatonin in maintaining gut health and microbiota balance, which are linked to sleep and circadian rhythm regulation.

Neuronal pathway involved in sleep regulation

The stress system: Hypothalamic-pituitary-adrenal axis

The hypothalamic-pituitary-adrenal (HPA) axis is crucial for regulating wakefulness and sleep patterns. Any disruption in this system, whether at the level of the corticotropin releasing hormone (CRH) receptor, glucocorticoid receptor (GR), or mineralocorticoid receptor, can lead to sleep disturbances (108). This is exemplified by a study showing that chronic corticosterone treatment in rats induced sleep disturbances through a defined neural circuit: downregulation of GR in the locus coeruleus (LC) led to activation of its noradrenergic neurons, which in turn inhibited GABAergic neurons in the ventrolateral preoptic area (VLPO). These results suggest that GR in LC may play a key role in sleep disorders (109). Conversely, sleep disruption can also impair the HPA axis function. A comprehensive study in rats demonstrated that chronic sleep restriction (CSR) induces decreased basal corticosterone and alters the timing of the corticosterone response peak (110), creating a potential feedback loop wherein sleep loss perpetuates neuroendocrine dysfunction. Compelling evidence has further illustrated that gut microbiota is a potential modulator of the HPA axis. Wu et al. found that serum corticosterone levels were more robustly increased after a transient social encounter in germ-free and antibiotic cocktail (ABX)-treated mice than in specific pathogen-free controls, and injection of the corticosterone synthesis blocker metyrapone (MET) or adrenalectomy (ADX) can abolish this phenomenon. The study demonstrated that gut bacteria can modulate the HPA stress response in mice and affect corticosterone production by the HPA axis (111). Strikingly, another recent study further found that the circadian rhythm of gut microbiota can modulate the suprachiasmatic nucleus (SCN) transcriptome and identified that the oscillation of Limosilactobacillus reuteri may modulate the rhythmicity of corticosterone (112). The rhythmicity of corticosterone is an important output of endogenous circadian signals which send signals to the brain to maintain the balance of sleep-wake cycle (31). Therefore, the gut microbiota may regulate the sleep-wake circadian rhythm through the HPA axis.

Notably, a recent clinical study gives complementary evidence to the role of the HPA axis in gut microbiota-brain communication in sleep disorders. A randomized controlled trial (RCT) reported that Bifidobacterium breve CCFM1025 can improve sleep quality by regulating the activity of the HPA axis (113). This provides direct interventional evidence that the gut microbiota-HPA axis—sleep pathway is operative in human sleep disorders.

Collectively, these studies implicate the potential role of the HPA axis in mediating the association between gut microbiota and sleep. The gut dysbiosis observed in sleep disorders may drive HPA axis dysregulation (e.g., GR overactivation), which can disrupt the balance of key sleep-wake circuits by increasing excitability of the LC and suppressing the VLPO, ultimately promoting a state of hyperarousal and the manifestation of sleep disturbances. Future research may include exploration of markers including salivary/serum cortisol (as a marker of the HPA axis), and heart rate variability (as a marker of the sympathovagal balance) to illustrate the underlying mechanism of gut-microbiota–brain interaction.

Immune pathways for gut microbiota and sleep interactions

The intestine is recognized as the body's largest immunological organ and is crucial for sustaining health. The intestinal vascular barrier serves a crucial protective function by obstructing the entry of harmful substances from the intestinal lumen into the bloodstream (114). Compromised barrier function is intricately associated with numerous intestinal and extraintestinal disorders (115). The gut microbiota is essential for sustaining immunological equilibrium by modulating metabolic and immune functions. Collectively, these mechanisms govern intestinal immune activity and sustain overall immunological homeostasis. Sang et al. have constructed a high-efficiency sleep deprivation model, elucidating for the first time the molecular mechanism by which sleep deprivation triggers cytokine storms and systemic inflammation via the PGD2/DP1 signaling axis (116). This discovery is significant for comprehending the relationship between sleep and immunity (116). The gut microbiota plays a crucial role in regulating immune responses, and its disruption can lead to systemic inflammation, potentially exacerbating conditions such as cytokine storms.

Gut microbiota can affect the permeability of the blood–brain and intestinal barriers, allowing intestinal immune cells and pathogens to access the central nervous system (117). The disruption of normal physiological functions in the gut microbiota causes damage to the intestinal lining and weakens the intestinal barrier. This disruption subsequently leads to immune system dysregulation, fostering the emergence of inflammatory diseases that can impact various organ systems (118). Simultaneously, a bidirectional connection exists between sleep and immune system function (119). Our prior research indicates that SD markedly elevates blood concentrations of proinflammatory cytokines (including TNF-α, IL-1β, and IL-6), while concurrently reducing levels of anti-inflammatory cytokines (such as IL-10) (120). Subsequent research indicates that insufficient sleep not only heightens the response to proinflammatory cytokines but also results in learning and memory impairments, which may be strongly associated with disturbances in the gut microbiota (121). Huynh et al. demonstrated that monocyte-derived TNF engages with Tnfrsf1a-expressing glutamatergic neurons in the thalamic lateral posterior nucleus to facilitate sleep, hence aiding in the restoration of heart function postmyocardial infarction and diminishing the likelihood of cardiovascular events (122). Moreover, increased TNF-α levels correlate with the severity of OSA (123). Animal studies indicate that OSA can elevate serum levels of LBP, IL-6, and TNF-α, while also modifying the composition of gut microbiota, such as decreasing Bifidobacterium levels (28). The intestinal microbiota of patients with obstructive sleep apnea experiences significant alterations, perhaps resulting in cognitive impairment through the activation of a systemic inflammatory response (124).

Our prior research indicates that sleep deprivation can induce local and systemic inflammatory responses in the gut by disrupting the gut microbiota ecosystem. Furthermore, FMT from sleep-deprived donors into germ-free mice resulted in hippocampal inflammation in the recipient mice (120). Further investigation indicates that acute sleep deprivation intensifies systemic inflammation and psychological disorders by disrupting gut microbiota and circadian rhythms (125). Probiotic fermentation products derived from germinated grains can markedly enhance sleep quality in mice, potentially linked to the modulation of neurotransmitter and inflammatory factor levels, the improvement of intestinal flora composition, and the elevation of SCFA concentrations (126). The intricate relationships among gut microbiota, the immune system, and sleep underscore their significance in health maintenance and offer novel perspectives for the treatment of associated disorders. Consequently, future research needs to investigate whether inflammatory substances in the peripheral gut might influence specific brain regions to regulate sleep.

Microbiota-targeted interventions for improving sleep

Gut microbiota is essential in regulating sleep and the gut-brain axis. Recent studies have highlighted the potential of microbiota-targeted interventions to offer therapeutic benefits in sleep and circadian homeostasis. Traditional treatments for sleep disorders often come with significant side effects, underscoring the need for alternative approaches. Microbiota-targeted therapies, including probiotics, prebiotics, synbiotics, and FMT (Figure 4), present promising strategies for improving sleep quality and managing sleep disorders may be promising novel strategies for the management of sleep disorders (Table 2).

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Probiotics

Probiotics are described as living microorganisms that give a health benefit when taken in suitable concentrations (127). The beneficial effects of probiotics in sleep occur through diverse mechanisms, including the regulation of neurochemical pathways such as the enhancement of GABA production, reduction of stress hormones like cortisol, and the regulation of serotonin pathways (128). Emerging clinical studies have demonstrated the role of specific probiotic strains (e.g., Lactobacillus and Bifidobacterium) in improving sleep across various populations. For example, Lactobacillus plantarum PS128 has been shown to improve sleep quality in chronic insomnia patients by enhancing delta power during N3 sleep, reflecting deeper and more restorative sleep (129). Additionally, Bifidobacterium breve CCFM1025 significantly reduced cortisol levels and improved subjective sleep quality in individuals with insomnia, pointing to the ability of probiotics to attenuate HPA axis hyperactivity (113). In parallel, college students under stress exhibited reduced insomnia symptoms and improved their sleep quality during exam periods after taking Lactobacillus plantarum JYLP-326 (130), further supporting the role of probiotics in mitigating stress-induced sleep disruptions.

Probiotics have also demonstrated sleep benefits in populations with neuropsychiatric disorders. Bifidobacterium animalis subsp. lactis Probio-M8 demonstrated improvements in sleep disturbances in patients with PD (131). Notably, Lactobacillus acidophilus has shown promise in patients with amphetamine-type stimulant use disorder, with greater reductions in PSQI scores observed in the probiotic group compared to the placebo group (132). This suggests that probiotics could have therapeutic potential for improving sleep disturbances related to substance use and withdrawal. Additionally, probiotics including Lacticaseibacillus paracasei 207-27 and Bifidobacterium adolescentis SBT2786 lengthened the objectively measured sleep duration in healthy participants (133, 134), underscoring the potential for probiotics to benefit both clinical and nonclinical populations.

Probiotics have demonstrated a considerable influence in enhancing sleep and regulating gut microbiota in experiments on animals. Specifically, supplementary Lacidofil demonstrated an enhancement in the length of nonrapid eye movement (NREM) sleep during the latter half of the photoperiod, contributing to improved sleep quality (135). Supplementation with Bifidobacterium animalis BB-12 can enhance sleep efficiency and diminish anxious behavior in rats (136). Supplementation with the Probiotic Fermented Germinated complex enhances sleep duration and diminishes anxious behavior in mice (126). Meanwhile, by blocking NF-κB signaling, Lactobacillus plantarum P72 and hemp seed oil efficiently increased 5-HTergic and GABAergic systems, reducing the symptoms of depression, anxiety, and insomnia in rats (137). Elevated doses of SG031 have demonstrated a protective effect in alleviating stress-induced sleep disorders, evidenced by a reduction in wakefulness and an increase in total sleep duration, alongside a decrease in depression-like behaviors and an enhancement of social interaction, which is crucial for improving sleep quality and social functionality (138).

Further study indicates that probiotics can mitigate intestinal barrier failure and neuroinflammation induced by SD while enhancing the intestinal microbiota in mice (139). The oral treatment of SLAB51 improves brain antioxidant capacity, modulates gut-brain axis hormones, and diminishes peripheral and cerebral inflammation resulting from CSR (140). Furthermore, treatment with Lactiplantibacillus plantarum 124 mitigated SD-induced intestinal oxidative stress, inflammation, and intestinal barrier dysfunction by modulating intestinal microbiota (141). Wang et al. showed that Faecalibacterium prausnitzi supplementation also enhances gut barrier integrity, reduces inflammation, and corrects biological disorders (142). These findings provide strong scientific support for the potential use of probiotics in regulating the gut-brain axis and improving sleep disorders. They also highlight promising avenues for future clinical research and the advancement of therapeutic strategies.

In conclusion, probiotics show promise as a potential intervention for improving sleep. However, most clinical evidence is derived from small-scale studies with significant heterogeneity in design. Future studies should focus on large-scale, double-blind, randomized trials to validate the therapeutic effects and incorporate animal models to uncover precise mechanisms, paving the way for targeted interventions.

Prebiotics

Prebiotics are substrates that are selectively utilized by host microorganisms conferring a health benefit (143). Recent clinical studies provide supportive evidence for the association between prebiotics and sleep regulation. In a randomized, double-blind, placebo-controlled study, partially hydrolyzed guar gum (PHGG) supplementation over 12 weeks has been revealed to significantly improve OSA-MA (Oguri–Shirakawa–Azumi Sleep Inventory, Middle-Aged version) scores in healthy elderly individuals (144). Similarly, resistant dextrin (RD) administered to females with type 2 diabetes mellitus led to favorable improvements in PSQI scores compared to placebo, highlighting its efficacy in populations with metabolic dysfunctions (145). In addition to the improvements in subjective sleep scales, prebiotics can also affect objective sleep indicators; for instance, yeast mannan supplementation significantly extended N3 sleep duration, shortened N3 latency, and increased total time in bed among individuals with defecation discomfort (146). Further work is needed to assess the therapeutic effect of prebiotics on sleep disorders.

Animal studies indicate that prebiotics may enhance sleep quality and ameliorate intestinal inflammation and barrier function. Dietary prebiotics enhanced NREM sleep by influencing particular metabolites of gut microbiota in rats (147). The relative abundances of Parabacteroides distasonis and Ruminiclostridium 5 were likewise elevated; moreover, Ruminiclostridium 5 and cholic acid were associated with core body temperature realignment cycles during light/dark reversal in rats (65). Bowers et al. discovered that a prebiotic diet extended NREM and REM sleep in rats, with the relative abundance of Parabacteroides distasonis potentially playing a significant role in facilitating the sleep-enhancing effects of the prebiotic diet (148). Chung et al. conducted further studies demonstrating that supplementation with particular prebiotics may enhance gut physiology, cognitive behavior, and motor performance affected by sleep loss through the modulation of inflammation and circadian rhythms (149). Collectively, the findings provide empirical evidence for the utilization of prebiotics as a potential approach to ameliorate sleep.

Synbiotics

The definition of a synbiotic is a mixture comprising live microorganisms and substrate(s) selectively utilized by host microorganisms that confers a health benefit on the host (150). Clinical studies highlight the potential of synbiotics in sleep improvement. Chan et al. (151) demonstrated that an intervention combining probiotics including Bifidobacterium and Lactobacillus, prebiotics such as inulin and oligosaccharides (OS), and postbiotic extracts significantly reduced PSQI scores after eight weeks in participants with sleep disturbances. Similarly, Lau et al. (2024a) showed that SIM01, a synbiotic formulation containing Bifidobacterium strains, alongside prebiotics like galactooligosaccharides (GOS), xylo-oligosaccharides (XOS), and resistant dextrin, alleviated insomnia symptoms in post-acute COVID-19 syndrome (PACS) patients (152). These studies demonstrate that synbiotics can effectively improve sleep quality. The study involving animals has demonstrated that the synbiotic combination of Faecalibacterium prausnitzii (ATCC-27766) with fructooligosaccharides (FOS) and galactooligosaccharides (GOS) can ameliorate treatment-resistant depression (TRD) symptoms in rats by enhancing intestinal health, mitigating neuroinflammation, modulating neurotransmitters, and altering intestinal microbial composition (153). Future efforts should focus on developing synbiotics tailored to specific sleep disorders, optimizing the synergistic and complementary interactions between probiotics and their substrates to maximize efficacy.

Comparative evidence and the viability of microbiota-targeted therapies

As of yet, no direct head-to-head randomized trials have evaluated microbiota-targeted therapies regarding sleep effects, such as probiotics compared to synbiotics or probiotics compared to FMT. The majority of research has assessed a singular intervention in comparison to placebo or standard treatment. Among these methodologies, probiotics have been the subject of substantial research, with numerous randomized controlled studies indicating enhancements in sleep quality and architecture, alongside favorable safety and accessibility, although individual responses may differ (113, 129). In terms of invasiveness, regulatory compliance, and standardization, probiotics and prebiotics seem most suitable for widespread clinical use, with synbiotics following closely after. FMT, in contrast, encounters significant obstacles such as donor screening, processing standardization, infection risk, and regulatory limitations, making it more suitable for research environments or specific refractory situations. Subsequent investigations ought to emphasize direct comparison trials and cost-effectiveness studies to elucidate the relative advantages, hazards, and therapeutic relevance of these microbiota-targeted therapies.

Investigating the sleep-microbiota-gut-brain axis: toward mechanisms and causality

The brain-gut axis's influence on sleep disorders has yet to be comprehensively explained (2). Early studies in the microbiome field were primarily correlative, identifying microbial communities associated with either healthy sleep patterns or sleep disorders. While these associations provided foundational insights, understanding how microbiota causally influences sleep and developing microbiome-based therapies requires systematic evaluation of a comprehensive evidence base. Given the growing number of studies in this area, it is challenging to assess each study's individual contributions to the broader understanding of microbiome-sleep interactions. Here, we propose a framework to evaluate the connection between microbiome and sleep disorders through a 4-tiered “funnel” approach, progressing from associative studies to molecular mechanistic investigations. This strategy facilitates a clearer understanding of how the microbiota drives sleep-related phenotypes and informs the development of targeted therapeutic interventions (Figure 5).

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Level 1: Initial associations identified between gut microorganisms and sleep. Associations between the gut microbiome and sleep disorders can be investigated through multimodal approaches. Neuroimaging techniques, such as functional magnetic resonance imaging and EEG, combined with sleep evaluations, cognitive assessments, actigraphy, and polysomnography (PSG), provide valuable insights into the physiological mechanisms underlying sleep and its impact on cognitive performance (159, 160). Concurrently, the collection of biological samples—including blood, saliva, urine, and feces—is essential for elucidating potential mechanistic pathways. Fecal samples serve as a reliable resource for examining the diversity and dynamic changes in the gut microbiota, laying a critical foundation for uncovering potential associations between alterations in the intestinal microbiome and sleep disorders (41). Level 2: Discovery of potential biomarkers linked to sleep disorders through the gut-brain axis. To identify potential biomarkers linking the gut microbiota to sleep disorders, advanced multi-omics approaches and computational tools are employed. Machine learning algorithms integrate 16S rRNA sequencing, metagenomic analyses, metabolomics, and clinical data to analyze large-scale datasets, enabling the classification of microbial signatures and functional pathways associated with sleep disturbances. Identified biomarkers, such as specific microbial strains, microbial-derived metabolites, or altered metabolic pathways, provide valuable targets for understanding sleep disorders and lay the groundwork for the development of personalized diagnostic and therapeutic strategies. Level 3: Establishing causality between gut microbiota and sleep disorders. To establish a causal relationship between gut microbiota and sleep disorders, rigorous experimental approaches are required. FMT serves as a pivotal tool, enabling the transfer of gut microbial communities from individuals with sleep disorders to germ-free or antibiotic-treated animal models, with the goal of identifying causative microbial strains that produce sleep phenotypes. Interventions based on the microbiome, demonstrated through RCTs and crossover studies, exhibit considerable potential for enhancing sleep quality and addressing sleep disorders. A longitudinal intervention study design, coupled with multiple sampling of the gut microbiome and machine learning methods, can yield crucial time series data to elucidate the effects of sleep disorders on microbial composition and function (161). Multimodal longitudinal design can be utilized in follow-up cohort studies to investigate the function of the brain-gut axis in the etiology of sleep disorders and the severity of their symptoms. Such studies can inform novel avenues of treatment for sleep disorders and disorders comorbid with sleep disorders. Level 4: Developing microbiome-based interventions to establish novel strategies for addressing sleep disorders. At this level, RCTs and crossover studies are employed to rigorously assess the therapeutic efficacy of microbiome-based interventions in ameliorating sleep disorders. These interventions may include specific microorganisms (e.g., probiotics) or their bioactive metabolites, such as SCFAs and other microbial-derived compounds. RCTs provide robust evidence by comparing intervention groups with placebo controls, while crossover designs allow within-subject comparisons, enhancing statistical power and minimizing variability. Sleep parameters, including architecture and quality, are evaluated using PSG, actigraphy, and subjective assessments. Additionally, neuroinflammatory markers, neurotransmitter levels, and gut microbial composition are analyzed to elucidate mechanisms underlying therapeutic effects. These studies are critical for translating microbiome research into effective, evidence-based interventions for sleep disorders. The funnel framework presented here offers a systematic approach to evaluate emerging microbiome-associated diseases, enabling the identification of microorganisms and microbial metabolites with consistent and reproducible effects on host physiology. These insights will drive the development of novel small-molecule therapies and cell-based interventions, advancing treatment strategies for microbiome-associated human diseases, including sleep disorders.