Probiotic VSL#3 Eases Long COVID Fatigue in Randomized Trial

Five years after the COVID-19 pandemic began, long COVID remains one of medicine’s most stubborn puzzles: millions of patients worldwide continue to struggle with persistent fatigue, cognitive difficulties, and impaired quality of life, yet no approved disease-modifying therapy exists. Now, a randomized, double-blind, placebo-controlled trial conducted in Milan suggests that a well-known probiotic formulation may offer meaningful relief. The DELong#3 trial, published in the British Journal of Biomedical Science, found that four weeks of supplementation with VSL#3, a high-concentration multi-strain probiotic, significantly reduced fatigue in patients with long COVID, with benefits that persisted even after treatment stopped.

The trial recruited patients from the Long COVID Outpatient Clinic at IRCCS Fondazione Ca’ Granda Ospedale Maggiore Policlinico between November 2022 and November 2023. Of 347 individuals screened, 53 met the strict inclusion criteria: documented SARS-CoV-2 infection, persistent symptoms lasting at least three months, and clinically relevant fatigue defined by a Chalder Fatigue Scale (CFS) score of at least 4 out of 11. After excluding five participants with low adherence, 48 patients completed the study, 25 in the placebo arm and 23 in the VSL#3 arm. The cohort was evenly split between sexes, with a median age of 54 years and a median symptom duration of 30 months, underscoring how entrenched these symptoms had become.

VSL#3 is no ordinary probiotic. Each sachet delivers 450 billion colony-forming units of eight live freeze-dried bacterial strains, including four Bifidobacterium and four Lactobacillus species along with Streptococcus thermophilus. Participants took two sachets daily for 28 days, with placebo sachets identical in appearance, smell, and taste. The choice of formulation was deliberate: several of its constituent genera, particularly Bifidobacterium and Lactobacillus, have been repeatedly reported as depleted in patients with COVID-19 and long COVID, and their loss has been linked to persistent inflammation and symptom burden.

The primary endpoint was unambiguous. Patients receiving VSL#3 experienced a 24.24 percent median reduction in CFS score, compared with just 6.06 percent in the placebo group, a difference that reached statistical significance at p = 0.037. The responder rate told an even clearer story: 68 percent of VSL#3-treated patients improved meaningfully, versus 35.7 percent of those on placebo (p = 0.019). Crucially, the clinical benefit was maintained at the four-week follow-up visit after treatment discontinuation, arguing against a purely transient effect of the supplementation period.

Beyond fatigue, the trial captured improvements across selected patient-reported outcomes. Quality-of-life measures from the SF-36 survey showed gains in physical functioning and role limitation due to emotional problems in the VSL#3 group, and gastrointestinal symptom scores improved as well, with notable reductions in epigastric pain. These parallel improvements align with the trial’s underlying hypothesis: that the gut, far from being a bystander in long COVID, may be an active participant in the disease process.

To probe the biological mechanisms, the researchers layered clinical outcomes with an ambitious multi-omic workup. Fecal samples underwent 16S rRNA gene sequencing to profile gut microbiota composition, while targeted metabolomics quantified short-chain fatty acids and neuromodulators such as dopamine and serotonin in both fecal and serum samples. Blood analyses included immune cell phenotyping by multiparametric flow cytometry and serum cytokine profiling using a 45-plex Luminex panel. The integration of these datasets allowed the team to look for coordinated patterns linking microbial changes, immune signaling, and clinical response.

The microbiota findings were selective rather than sweeping. Overall microbial diversity, measured by alpha and beta diversity indices, did not differ significantly between groups at baseline or after treatment. Yet differential abundance analysis revealed that VSL#3 supplementation enriched specific health-associated taxa, including Bifidobacterium, Lactobacillus, Streptococcus, and Barnesiella, several of which correspond to strains in the administered formulation. Notably, clinical responders showed a distinct microbial signature beyond the supplemented organisms, with enrichment of commensal genera such as Bacteroides, Ruminococcus, Coprococcus, Phascolarctobacterium, Akkermansia, and Clostridium cluster IV, taxa associated with short-chain fatty acid production and gut ecosystem balance. Functional prediction analysis also pointed to enrichment of glycine, threonine, and methionine metabolism pathways in the VSL#3 group.

On the immune front, the results were more cautious. Within the VSL#3 group, circulating levels of IL1α, IL6, IL7, IFNα, and G-CSF declined from baseline to the end of treatment, hinting at a partial attenuation of the persistent low-grade immune activation characteristic of long COVID. However, these cytokine changes did not reach statistical significance when compared directly with the placebo arm, and the authors emphasize they should be interpreted as exploratory, hypothesis-generating observations. Immune cell phenotyping did reveal remodeling of peripheral populations, with increased proportions of CD8-positive T cells and CD68-positive macrophages alongside reduced CD66b-positive granulocytes, suggesting a shift in innate and adaptive immune balance following supplementation.

Perhaps the most intriguing findings emerged from correlation analyses linking the three data layers. In responder patients, the bacterial genus Coprococcus showed positive associations with the anti-inflammatory cytokine IL10 and with IL7, while isovaleric acid correlated positively with Coprococcus and valeric acid with Ruminococcus. Conversely, in non-responders, Akkermansia showed negative correlations with IL17 and G-CSF. These coordinated microbiota-immune-metabolic patterns, the authors suggest, may reflect broader interaction networks relevant to gut-brain communication, the axis increasingly implicated in fatigue and neurocognitive symptoms across long COVID and related post-infectious conditions such as myalgic encephalomyelitis/chronic fatigue syndrome.

The study’s strengths include its randomized, double-blind, placebo-controlled design and the depth of its integrated analyses, but the authors are candid about limitations: the small cohort size, the short post-treatment follow-up, the exploratory nature of the multi-omic work, and the inability to resolve whether enriched taxa represent durable microbiota remodeling or transient detection of supplemented strains. Longer-term, strain-resolved shotgun metagenomic studies will be needed to settle that question. Still, the persistence of clinical benefit four weeks after treatment stopped, combined with the enrichment of commensal taxa not present in the formulation, offers a compelling signal. As the first randomized placebo-controlled evidence supporting microbiota-targeted intervention for long COVID fatigue, the DELong#3 trial opens a concrete path for larger trials that could finally translate gut-directed therapy from hypothesis to standard of care.

Subject of Research: A randomized placebo-controlled trial of VSL#3 probiotic supplementation for fatigue in long COVID patients, with integrated microbiota, immune, and metabolomic analyses

Article Title: VSL#3® supplementation improves fatigue in long COVID: results from the DELong#3 randomized placebo-controlled trial

Article References: Amoroso, C., Marinoni, B., Maragno, P., Rimondi, A., Bottaro, F., Ciafardini, C., Muià, M., Honcharyuk, I., Noviello, D., Caridi, B., Sommella, E. M., Bandera, A., Gori, A., Mantero, M., Blasi, F., Ferrucci, R., Priori, A., Strati, F., Vecchi, M., … Caprioli, F. (2026). VSL#3® supplementation improves fatigue in long COVID: results from the DELong#3 randomized placebo-controlled trial. British Journal of Biomedical Science, 83, Article 16993. https://doi.org/10.3389/bjbs.2026.16993

Image Credits: AI Generated

DOI: 10.3389/bjbs.2026.16993

Keywords: long COVID, VSL#3, probiotics, gut microbiota, fatigue, randomized controlled trial, immune dysregulation, microbiota-gut-brain axis, Chalder Fatigue Scale, short-chain fatty acids, Bifidobacterium, metabolomics