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Article

The Effect of Ergothioneine Supplementation on Cognitive Function, Memory, and Sleep in Older Adults with Subjective Memory Complaints: A Randomized Placebo-Controlled Trial

by
Ian T. Zajac
1,2,
Naomi Kakoschke
1,
Barbara Kuhn-Sherlock
3 and
Linda S. May-Zhang
4,*
1
Health & Biosecurity, Commonwealth Scientific and Industrial Research Organization, Adelaide, SA 5000, Australia
2
College of Education, Psychology and Social Work, Flinders University, Adelaide, SA 5042, Australia
3
BKS Consulting, Hamilton 3216, New Zealand
4
Blue California, Rancho Santa Margarita, CA 92688, USA
*
Author to whom correspondence should be addressed.
Nutraceuticals 2025, 5(3), 15; https://doi.org/10.3390/nutraceuticals5030015 (registering DOI)
Submission received: 9 May 2025 / Revised: 18 June 2025 / Accepted: 24 June 2025 / Published: 27 June 2025
(This article belongs to the Special Issue The Role of Nutraceuticals in Central Nervous System Disorders)
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Abstract

Ergothioneine is a diet-derived antioxidant with emerging evidence of neuroprotective benefits, but no dose-ranging study has evaluated its effects in healthy older adults. In this 16-week randomized, double-blind, placebo-controlled trial, 147 adults aged 55–79 with subjective memory complaints received ergothioneine (10 mg or 25 mg/day ErgoActive®) or placebo. The primary outcome was the change in composite memory (CNS Vital Signs). Secondary outcomes included other cognitive domains, subjective memory and sleep quality, and blood biomarkers. At baseline, participants showed slightly above-average cognitive function (neurocognitive index median = 105), with plasma ergothioneine levels of median = 1154 nM (interquartile range = 889.9). Plasma ergothioneine increased by ~3- and ~6-fold for 10 mg, and ~6- and ~16-fold for 25 mg, at weeks 4 and 16, respectively (p < 0.001). 25 mg ergothioneine showed a within-group improvement in composite memory at week 4 (p < 0.05), although this was not sustained. Reaction time improved in both groups, dependent on time. Other domains showed null or limited effects. Subjective prospective memory and sleep initiation improved dose-dependently, with significant effects at 25 mg (p < 0.05). Liver function improved and a within-group increase in telomere length was noted. In conclusion, ergothioneine supplementation was safe and well tolerated, with evidence suggesting some benefits in this cohort of healthy older adults. Longer trials in individuals with lower baseline ergothioneine or cognitive function are warranted.

1. Introduction

Age-related cognitive decline is a growing public health concern with substantial implications for individuals, caregivers, and healthcare systems. Cognitive deterioration impairs quality of life, compromises independence, and increases long-term care needs [1,2]. Although some decline in cognitive function is expected with age, the extent varies considerably among individuals [3,4]. Since cognitive decline is influenced by modifiable risk factors [5,6,7], identifying effective interventions is a critical priority for aging populations.
Among the modifiable lifestyle factors, nutrition plays an important role in maintaining cognitive health. Epidemiological studies have linked specific dietary patterns, such as the Mediterranean diet, Dietary Approaches to Stop Hypertension (DASH) and the Mediterranean–DASH Intervention for Neurodegenerative Delay (MIND) diets, with slower cognitive decline and reduced risk of neurodegenerative diseases [8,9,10,11,12,13]. However, long-term adherence to these comprehensive dietary approaches may pose practical challenges for older adults, especially those experiencing appetite changes, sensory impairments, or limited access to fresh food [14]. This has led to a growing interest in targeted, single-nutrient interventions that leverage bioactive compounds with known neuroprotective mechanisms. Nutrients such as B vitamins, antioxidants (e.g., vitamins C and E, flavonoids, polyphenols), and omega-3 fatty acids have been studied for this purpose, although the findings have been inconsistent [15,16,17,18,19], potentially due the low bioavailability, rapid metabolism, or a lack of standardized dosing.
One such compound gaining increasing attention is ergothioneine, a naturally occurring amino acid antioxidant found predominantly in mushrooms and minorly in other foods, including oats, beans, nuts, and meat products [20,21,22]. As far as we know, ergothioneine is synthesized only by certain bacteria, fungi and yeasts [22]. Higher plants appear to obtain ergothioneine by their roots interacting with fungi and bacteria in soil [23]. Although not synthesized in the human body, ergothioneine is efficiently absorbed via the OCTN1 transporter (also known as the ergothioneine transporter, or ETT), which is expressed in many tissues, including the intestine, red blood cells, kidneys, bone marrow, immune cells, skin, and brain [24,25]. This transporter enables ergothioneine to accumulate in high concentrations in organs vulnerable to oxidative stress and inflammation. Ergothioneine has multiple cellular protective functions, including scavenging reactive oxygen species, chelating redox-active metals, suppressing pro-inflammatory signaling, and protecting mitochondrial function [26]. In aged mouse models, ergothioneine supplementation improves learning and memory by preserving hippocampal neurogenesis and supporting neuronal maturation [27,28,29]. Recent evidence shows that ergothioneine enters mitochondria and improves mitochondrial function [30,31,32]. In humans, pharmacokinetic studies indicate that ergothioneine is rapidly absorbed, retained in the body with significant elevations in plasma and whole blood, and minimally excreted in urine [33].
A growing body of evidence in humans suggests a protective role of ergothioneine against cognitive decline and supporting cognitive function. Multiple correlative studies in older adults have associated lower blood plasma levels of ergothioneine with mild cognitive impairment (MCI) [34,35,36], dementia [37,38], Parkinson’s disease [39], and cerebrovascular disease [36,38]. Notably, a study by Wu et al. [36] found that low plasma ergothioneine predicted faster rates of cognitive decline across multiple cognitive domains over five years. Despite these consistent associations, interventional studies have remained limited. A recent placebo-controlled pilot study in older adults with MCI showed that 25 mg ergothioneine, taken three times per week for one year, improved learning performance and stabilized the neurofilament light chain, a marker of neuronal injury [40]. However, whether ergothioneine supplementation benefits cognitively healthy older adults without diagnosed cognitive impairments remains unknown.
The present study aimed to investigate the effects of ergothioneine supplementation on cognitive function in healthy adults aged 55 to 79 years with subjective memory complaints. We conducted a 16-week randomized, double-blind, placebo-controlled trial with three arms: 25 mg ergothioneine, 10 mg ergothioneine, and placebo. The primary objective was to assess changes in compositive memory using the Central Nervous System Vital Signs (CNS-VS) computerized neurocognitive test battery. A number of secondary outcomes were explored, including other CNS-VS cognitive domains, subjective memory, subjective sleep quality, and blood biomarkers related to inflammation and safety.

2. Materials and Methods

2.1. Study Design, Protocol Deviations, and Ethics Approval

This single-center, 16-week, randomized, double-blind, placebo-controlled trial was conducted at the Commonwealth Scientific and Industrial Research Organization (CSIRO) Clinical Research Unit in Adelaide, South Australia. The study was conducted in accordance with the principles of the Declaration of Helsinki and requirements of Good Clinical Practice. The study was approved by the CSIRO Human Research Ethics Committee (2021_015_HREC) and pre-registered in the Australian and New Zealand Clinical Trials registry (ACTRN12621001607864). The registry listed two primary cognitive outcomes: the CNS Vital Signs (CNS-VS) neurocognitive battery and the Composite Autonomic Symptom Score (COMPASS) questionnaire. However, due to protocol amendment to select for one primary outcome, the final analysis focused on only the CNS-VS composite memory domain as the primary outcome and excluded the COMPASS as a primary outcome. The remaining CNS-VS domain scores were analyzed as secondary outcomes. These changes were finalized in a statistical analysis plan in May 2023 (during patient screening) and are reported transparently here.
Informed consent was obtained from all participating subjects during the screening visit and before all the study procedures.

2.2. Participants, Screening, and Inclusion/Exclusion Criteria

Participants were community-dwelling males and females aged 55 to 79 with self-reported memory complaints. The inclusion criteria included a score of ≥25 on the Memory Assessment Clinics Questionnaire (MAC-Q) [41], indicating subjective memory concerns, and the absence of dementia as determined by the Montreal Cognitive Assessment [42]. Eligible individuals had a body mass index (BMI) between 18.5 and 35 kg/m2, normal or corrected-to-normal vision, no color blindness, access to email, and the capacity to provide informed consent.
The exclusion criteria included uncontrolled hypertension (average systolic blood pressure (BP) > 155 mmHg or diastolic BP > 100 mmHg), cognitive impairment (MoCA ≤ 22), current depression (GDS-15 ≥ 6), recent participation in other clinical trials, and any condition or treatment likely to affect cognitive function or study compliance (e.g., neurodegenerative disease, untreated sleep disorders, history of chemotherapy or radiotherapy, use of sedative medications, excessive alcohol intake, or high caffeine or mushroom consumption). The full inclusion and exclusion criteria are provided in Supplementary Materials Table S1.

2.3. Randomization, Blinding, and Intervention

Participants were randomly assigned to one of three arms: (1) 25 mg ErgoActive® L-ergothioneine (ERG), (2) 10 mg ErgoActive® L-ergothioneine, or (3) placebo at a 1:1:1 allocation. Randomization was performed using an Interactive Web-Based Randomization Service managed independently by the Adelaide Health Technology Assessment. Stratification was based on sex, age group (55–63.9, 64–70.9, 71–79.9 years), and MAC-Q score (25–27 or 28–30). Both the participants and researchers were blinded to the group allocation until after data analysis was complete.
ErgoActive® is a nature-identical form of L-ergothioneine (Blue California, Rancho Santa Margarita, CA, USA) with FDA Generally Recognized As Safe (GRAS) status (GRN#734). The investigational products were manufactured by a Current Good Manufacturing Practices (cGMP)-certified facility (ProTab Laboratories, Foothill Ranch, CA, USA). Ergothioneine capsules (10 mg or 25 mg) and identical placebo capsules (containing maltodextrin, microcrystalline cellulose, magnesium stearate, and silicon dioxide) were delivered in sealed, opaque containers. Participants were instructed to take one capsule daily with food in the morning for 16 weeks. Compliance was monitored throughout the 16-week intervention period. Those who were determined to have fallen below 80% compliance were followed up via phone call by site study staff. Participants were instructed to return any remaining product to the study site for accountability purposes.

2.4. Study Procedures

Participants were recruited via the CSIRO volunteer database, media ads, and targeted social media campaigns. Initial eligibility was assessed via a Research Participant Recruitment Application, followed by telephone screening. Eligible participants attended a screening visit, completed an informed consent form, and completed the CNS-VS cognitive test for familiarization to minimize practice effects [43].
The primary objective was to determine the effect of ergothioneine supplementation on cognitive performance assessed via the CNS-VS, which assessed 12 clinical domains, including a compositive memory score. The primary endpoint included the difference from baseline (day 0) to day 112 in compositive memory.
The CNS-VS is a computerized, individually administered and standardized neuropsychological battery designed for measuring and monitoring cognitive change over time [44]. The CNS-VS assessed composite memory, verbal memory, visual memory, processing speed, executive function, psychomotor speed, reaction time, complex attention, simple attention, motor speed, and cognitive flexibility (see Supplementary Materials Table S2 for descriptions of each domain). Standardized scores, which have a mean of 100 and a standard deviation of 15 (similar to IQ scores), are used to depict individual performance, with higher scores indicating better performance. Scores > 110 are defined as high function and high capacity, 90–110 are defined as normal function/capacity, 80–90 are defined as slight deficit and slight impairment, 70–79 are defined as moderate deficit and possible impairment, and <70 are defined as deficit and likely impaired. Participants completed the CNS-VS at baseline, week 4 and again at the endpoint (week 16).
The secondary objectives included the effects of ergothioneine supplementation on other CNS-VS cognitive domains, subjective memory, subjective sleep quality, and blood biomarkers related to inflammation and safety. Subjective memory was evaluated using the validated Prospective and Retrospective Memory Questionnaire (PRMQ), which measures the frequency of different types of memory failures [45]. The 16-item PRMQ contains 8 items representing prospective memory (both short term and long term, self-cued and environmentally cued) and 8 items representing retrospective memory (both short term and long term, self-cued and environmentally cued) and rated on a 5-point Likert scale. Subjective sleep quality was assessed using the Leeds Sleep Questionnaire (LSEQ), a 10-item visual analog scale that pertains to the ease of getting to sleep, quality of sleep, awakening from sleep, and behavior following wakefulness [46]. Both were administered at baseline, week 4 and the endpoint.
Biochemical markers were analyzed at a National Association of Testing Authorities Australia Laboratory (biochemistry, hematology, high-sensitivity C-reactive protein [CRP]) and the CSIRO Human Health analytical laboratory (ergothioneine levels, inflammatory markers, telomere length). Blood samples (~34 mL total) were collected via venipuncture after 10 to 16 h of fasting. Serum and plasma samples were processed under standardized conditions and stored at −80 °C. Cytokines (IL-1β, IL-6, IL-10, TNF-α, IFN-γ) were analyzed using a multiplex test kit (Merck, Darmstadt, Germany) on a Luminex 200 plate reader (Luminex, TX, USA). Ergothioneine levels were analyzed in ethylenediaminetetraacetic acid (EDTA) plasma by liquid chromatography quadrupole time-of-flight (LC-QToF) using a modification to the method of Cheah et al. [33]. Telomere length was analyzed in lymphocytes using flow cytometry. Trimethylamine N-oxide (TMAO) was analyzed post hoc using a competitive enzyme-linked immunosorbent assay (ELISA) kit (MyBioSource, San Diego, CA, USA) in plasma.

2.5. Sample Size and Statistical Analyses

A sample size (N = 150, 50 per group) was determined to detect a small-to-medium effect size (f = 0.14) for the primary outcome, with >90% power. Accounting for a 20% attrition rate, a final sample size of N = 120 would still retain >80% power (G*Power 3.1, α = 0.05, r = 0.5).
The primary analysis included the intention-to-treat (ITT) population. A per protocol (PP) analysis included those with full compliance (≥80%) and complete data. The ITT population included all participants with baseline (day 0) measurements who were randomized to one of the three intervention arms and had consumed at least one dose of the study product. The PP population included all participants who adhered to the major criteria in the protocol and attended all three visits within the required window with complete data for the primary outcome (CNS-VS) and at least 80% intervention compliance.
Mixed-effects repeated measures analysis of variance (ANOVA) was used for the primary and secondary outcomes, with treatment group, time point, sex, and interactions as fixed effects, age and Memory Assessment Clinics Questionnaire (MAC-Q) score as covariates, and subject as a random effect. Normality of residuals was visually assessed (e.g., histograms, Q-Q plots), and normalizing transformations were applied when necessary. Due to the extreme variability, the CNS-VS Simple Attention domain could not be reliably transformed and was excluded from the analysis. Pairwise comparisons were conducted using Tukey’s honestly significant difference (HSD) test. Within-group significance was determined if the 95% confidence interval (CI) range did not contain the value zero [47,48]. Secondary endpoints were analyzed using a similar mixed-model ANOVA approach, with the Benjamini–Hochberg 10% false discovery rate (FDR) procedure applied to significant interactions. The results are presented as least-squares means (LSM) with 95% Tukey-adjusted CIs. p-values of 0.05 or less were considered significant.

3. Results

3.1. Participant Characteristics and Recruitment

Baseline characteristics of the study population are presented in Table 1. Across all the groups, approximately 73% of participants in each group were female, with a median age of 69 years. Screening occurred from April 2022 to February 2023 (see Figure 1 for the CONSORT flow diagram). Although the trial originally aimed to enroll 150 participants, recruitment concluded at 147 due to timeline constraints and concerns related to the COVID-19 pandemic. Seven participants (4.8%) withdrew before completing the study, and thirteen participants (8.8%) did not meet the PP criteria.

3.2. Plasma Ergothioneine Levels

At baseline, plasma ergothioneine concentrations did not differ significantly across the groups, with median values of 1099.1 nM (placebo), 1188.1 nM (ERG 10 mg), and 1087.8 nM (ERG 25 mg) (Supplementary Materials Table S3). Mixed-model analyses showed significant main effects of treatment and time in both the ITT and PP samples (all p < 0.001). Pairwise comparisons revealed significant differences among all the groups (p < 0.001), confirming a dose- and time-dependent increase in plasma ergothioneine following supplementation. Within-group analyses showed significant increases in plasma ergothioneine at both week 4 and week 16 in the ERG 10 mg and ERG 25 mg groups (all p < 0.05). The ERG 10 mg group exhibited approximately a 3-fold increase at week 4 and a 6-fold increase at week 16, while the ERG 25 mg group showed ~6-fold and ~16-fold increases at the same time points (Figure 2).

3.3. Primary Outcome: Composite Memory Measured by CNS-VS Composite Memory Score

Cognitive function at baseline was comparable across groups for all the domains (Supplementary Materials Table S4). The subjects were considered on the slightly higher end of normal/average range in terms of cognitive function at baseline, as indicated by the Neurocognitive Index (NCI), a composite score used to describe a person’s global cognitive function. Median NCI scores were placebo 105 (IQR = 8.0,) ERG 10 mg 105.5 (IQR = 10.0), and ERG 25 mg 103.0 (IQR = 10.0).
The primary outcome, the Composite Memory score from the CNS-VS battery, was evaluated at weeks 4 and 16 in the ITT population. While we did not find any significant treatment or time effects, there were positive trends. At week 4, the 25 mg group showed a significant improvement from baseline (LSM = 5.20, 95% CI: 1.28 to 9.11), suggesting an early benefit in terms of memory performance. However, this effect was not maintained at week 16 (LSM = 0.22, 95% CI: –3.70 to 4.14). The 10 mg group showed no significant changes, while the placebo group showed modest, non-significant improvements at week 4 (LSM = 2.12, 95% CI: –1.97 to 6.20) and week 16 (LSM = 3.25, 95% CI: –0.85 to 7.35) (Figure 3A).
The findings were consistent in the PP population, where the 25 mg group again showed the greatest improvement at week 4 (LSM = 6.4, 95% CI: 2.2 to 10.6), which attenuated by week 16 (LSM = 0.2, 95% CI: −4.0 to 4.4). The lack of significant between-group differences was likely due to individual variability and limited statistical power.

3.4. Secondary Outcome: Cognitive Function Measured by CNS-VS (All Domains)

Analysis of between-group changes revealed a significant time-by-treatment interaction for Reaction Time in both the ITT (p = 0.02) and PP (p = 0.01) samples (Figure 3B). While the ERG 10 mg group showed modest improvements relative to placebo and ERG 25 mg at week 4, the ERG 25 mg group showed slightly greater improvements at week 16. An overall treatment effect was observed for Psychomotor Speed in the PP population (p = 0.04), driven by a significant difference between the ERG 10 mg and placebo groups (p = 0.02), favoring placebo (Figure 3C). However, none of the other pairwise comparisons for the other domains reached statistical significance.
Post hoc within-group analyses revealed domain-specific cognitive improvements from baseline over the 16-week intervention period (Supplementary Materials Table S5). Analyses reported in the text were from the ITT samples, although both the ITT and PP results are reported in Table S5. In the placebo group, significant improvements (p < 0.05) were observed in NCI, psychomotor speed and verbal memory at 16 weeks compared to baseline, likely reflecting practice effects associated with repeated testing. Participants receiving 10 mg ergothioneine demonstrated significant (p < 0.05) within-group increases in complex attention, cognitive flexibility, and executive functioning at 16 weeks compared to baseline. However, a significant decline was noted in visual memory, indicating a domain-specific decrement at this dose. The ERG 25 mg group showed a broader pattern of improvement, with significant (p < 0.05) changes observed in NCI, complex attention, cognitive flexibility, executive functioning, and verbal memory at 16 weeks. No significant decline was observed in visual memory for this group.

3.5. Secondary Outcome: Subjective Memory

Baseline scores on the PRMQ were comparable across groups (Supplementary Materials Table S4). Median PRMQ scores were placebo 40.0 (IQR = 12.0,) ERG 10 mg 39.0 (IQR = 12.0), and ERG 25 mg 40.0 (IQR = 8.0), indicating that the majority of participants reported mild to moderate subjective memory complaints but still within normal range.
A significant overall treatment effect was found for Prospective Memory in the PP sample (p = 0.03), with a trend in the ITT sample (p = 0.06). In the PP analysis, the placebo group showed a significant within-group decline (LSM: −1.26; 95% CI: −2.02 to −0.50), while no change was observed in the ERG 25 mg group (LSM: 0.05; 95% CI: −0.68 to 0.77). A direct comparison confirmed a significant difference between placebo and ERG 25 mg (p = 0.04), indicating a protective effect of 25 mg ergothioneine against subjective prospective memory decline over 16 weeks. Figure 4 plots the average change in Prospective Memory for the three treatment groups across the time points. No other significant interactions were observed for the PRMQ outcomes.

3.6. Secondary Outcome: Subjective Sleep Quality

Participants were well-matched for sleep quality at baseline, with no significant differences across groups in any LSEQ domains (Supplementary Materials Table S4). A significant overall treatment effect for the “Getting to Sleep” domain was observed in the PP sample (p = 0.04), with a trend in the ITT sample (p = 0.08). This effect was dose-dependent but primarily driven by greater improvements in the ERG 25 mg group relative to the placebo (Figure 5). Within-group analysis showed a significant improvement in the ERG 25 mg group (PP LSM: 3.27; 95% CI: 0.39 to 6.14), while the placebo group showed no significant change. These findings suggest that the higher dose ergothioneine may support sleep initiation, potentially through calming or neuromodulatory mechanisms. No other LSEQ domains showed significant treatment effects or interactions.

3.7. Exploratory Biomarker: Leukocyte Telomere Length

Baseline telomere length did not differ significantly between groups. No significant between-group differences were observed at week 16. However, within-group analysis revealed a significant increase in telomere length in the ERG 10 mg group (ITT LSM: 0.26; 95% CI: 0.02 to 0.49), with this effect primarily observed among female participants (ITT LSM: 0.37; 95% CI: 0.12 to 0.62) (Supplementary Materials Figure S1). Although exploratory, these findings suggest a potential dose- and sex-specific effect of ergothioneine on supporting telomere length in humans.

3.8. Inflammation

TNF-α showed a borderline treatment effect in the PP sample (p = 0.06), driven by a near-significant difference between the ERG 25 mg and placebo groups at week 16 (p = 0.07), and a significant within-group reduction in the ERG 25 mg group (p = 0.05). The other inflammatory markers (IFN-γ, IL-1β, IL-6, IL-10) showed no significant treatment effects.

3.9. Safety, Liver Function and TMAO Levels

Baseline plasma chemistry and hematology were comparable across groups (Supplementary Materials Tables S6), except for minor clinically insignificant differences in blood urea nitrogen and eosinophils.
Significant treatment effects were observed for creatinine, hemoglobin, and monocytes (Supplementary Materials Table S7). For creatine, an overall treatment effect was observed in both the ITT (p = 0.02) and PP (p = 0.01) populations. Participants in the ERG 10 mg group showed a slight reduction in creatinine levels (ITT LSM: −0.63; 95% CI: −2.02 to 0.7; PP: −1.18; 95% CI: −2.60 to 0.25), while the placebo group showed a modest increase (ITT LSM: 1.92; 95% CI: 0.45 to 3.40; PP: 2.04; 95% CI: 0.62 to 3.47). For hemoglobin, the overall model reached significance in the ITT analysis (p = 0.04). Small decreases were observed in both ergothioneine groups (e.g., ERG 10 mg ITT LSM: −1.91; 95% CI: −3.35 to −0.47), while the placebo group remained unchanged. The monocyte counts also differed significantly between groups, with a main treatment effect (p = 0.03) observed in both the ITT and PP samples. The ERG 10 mg group showed a slight increase (LSM: 0.034; 95% CI: 0.011 to 0.058), while the ERG 25 mg and placebo groups did not change. Although statistically significant, all changes remained within normal clinical reference ranges and were not considered clinically meaningful.
Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assessed as indicators of liver function. For AST, a significant treatment-by-time interaction was observed in both the ITT and PP populations (p = 0.03; Supplementary Materials Figure S2A). AST levels trended downward in both ergothioneine groups by week 4, and a significant within-group reduction was seen in the 25 mg group at week 16 (PP LSM: −1.43; 95% CI: −2.45 to −0.42). For ALT, a significant treatment-by-time-by-sex interaction was found in both the ITT (p = 0.02) and PP (p = 0.04) analyses, along with a trend toward a treatment-by-time interaction in the ITT population (p = 0.10; Supplementary Materials Figure S2B). By week 16, a dose-dependent between-group difference emerged (placebo vs. 25 mg: p = 0.03), driven by sex-specific effects. Among the male participants, ALT increased significantly in the placebo group (ITT LSM: +5.56; 95% CI: 2.55 to 8.57) but declined slightly in the 25 mg ergothioneine group (ITT LSM: −2.10; 95% CI: −5.83 to 0.21), with a significant between-group difference (placebo vs. 25 mg; p = 0.02). These findings suggest potential dose-dependent hepatoprotective effects of ergothioneine, particularly in males.
TMAO levels were measured to address potential concerns that microbial metabolism of ergothioneine might generate trimethylamine (TMA) [49], a precursor of TMAO, which is associated with cardiovascular risk [50,51]. Ergothioneine supplementation at 10 mg or 25 mg did not increase plasma TMAO levels (Supplementary Materials Figure S3). Importantly, no adverse events or unintended effects related to the investigational product were reported, supporting the safety and tolerability of ergothioneine supplementation.

4. Discussion

This single-center, randomized, double-blind, placebo-controlled trial investigated the effects of ergothioneine supplementation on cognitive function, memory and sleep in healthy older adults. The study expands on the limited body of interventional research by evaluating the effects of daily supplementation with 10 mg or 25 mg of ergothioneine over a 16-week period in individuals with subjective memory complaints but without diagnosed MCI, neurological disorders, or sleep disturbances.
Supplementation led to a dose-dependent plasma accumulation of ergothioneine, with 25 mg for 4 weeks yielding a ~6-fold increase, similar to 10 mg taken for 16 weeks. This mirrors pharmacokinetic findings in healthy young males, where plasma accumulation occurred within one week of supplementation [33]. Epidemiological studies have associated higher plasma ergothioneine levels with reduced risk of neurodegenerative diseases, mortality and cardiovascular disease [52]. Based on data compiled from patients with a range of disorders, including age-related macular degeneration, Parkinson’s disease, MCI, Alzheimer’s disease, and vascular dementia, Halliwell et al. proposed a hypothetical plasma threshold of ~810 nM to distinguish ‘healthy’ from ‘unhealthy’ individuals [22]. A large longitudinal aging study further found that each standard deviation increase in plasma ergothioneine was associated with a 25% reduction in the mortality risk [53]. Despite excluding subjects who consumed more than one serving of mushrooms per week, the baseline plasma levels of ergothioneine in our participants (median 1154 nM; IQR 887.9 nM) exceeded the proposed threshold of ~810 nM for ‘healthy.’ This suggests that a large subset of participants already had potentially ‘healthy’ levels of ergothioneine at baseline, obtained from dietary sources other than mushrooms.
For the primary outcome, composite memory scores improved in the 25 mg group at week 4, but this was not sustained and did not differ from the placebo. Reaction time scores improved modestly in both ergothioneine groups without a clear dose-response. Several secondary cognitive domains, including complex attention, cognitive flexibility, and executive functioning, showed within-group improvements at both doses but not significantly more than the placebo. Visual memory and psychomotor speed scores improved in the placebo group, suggesting potential practice or expectancy effects. A decline in visual memory in the 10 mg group remains unexplained. Overall, the objective cognitive effects of ergothioneine supplementation were considered modest and variable in this cognitively healthy cohort.
The participants had cognitive scores on a slightly higher end of the normal/average range, despite scoring >25 on the MAC-Q for memory complaints. Combined with variable baseline plasma ergothioneine levels by which the median was considered potentially ‘healthy’, this may have limited the potential to detect further cognitive improvement due to ceiling effects or contributed to the response variability. In addition, prior dietary intervention reviews suggest longer durations (e.g., 2–5 years) are often required to detect robust cognitive changes [54,55], suggesting that the 16-week intervention may have been insufficient.
Notably, the participants experienced dose-dependent improvements in subjective prospective memory, significant at 25 mg, while those on the placebo declined. Subjective memory assessments are increasingly recognized as markers of early cognitive change and strong predictors of future decline, particularly when persistent or accompanied by concern [56,57,58]. Our results align with findings from a recent pilot study, in which 25 mg ergothioneine taken three times weekly increased the Z-scores in Rey Auditory Verbal Learning Test assessments, which evaluate learning ability and memory in older adults with MCI following one year of supplementation [40]. While the objective cognitive effects were modest in this healthy, high-functioning cohort, improvements in subjective memory suggest early preventive potential.
Subjective sleep initiation also dose-dependently improved, particularly in the 25 mg group. These findings are consistent with previous studies in humans [59] and animal models, which reported that ergothioneine supplementation improved objective and subjective sleep measures [60]. Sleep disturbances are known contributors to cognitive decline and Alzheimer’s disease [56,57,58]. Refs. [61,62,63] suggest that subjective sleep assessments, similar to cognitive assessments, can be as meaningful as objective measures [64], particularly for detecting early changes or treatment responses in healthy or subclinical populations. Similar patterns have been reported with L-theanine supplementation, where improvements were consistently observed in subjective sleep quality, even in the absence of consistent objective changes [65]. Although sleep was an exploratory secondary outcome in this trial, the improvements in sleep initiation, along with prior findings, suggest that ergothioneine may modulate sleep-related pathways relevant to cognitive resilience.
We also investigated telomere length as a marker of cellular aging. Ergothioneine has been shown to slow telomere shortening and transiently increase telomerase activity in human fibroblasts under oxidative stress [66], and higher plasma ergothioneine has been linked to longer telomeres in population-based studies [67]. Our study is the first to investigate the effect of ergothioneine supplementation on telomere length. We found that telomere length increased significantly in the 10 mg group, particularly among females, although between-group differences were not observed. While inconclusive, these results contribute to growing interest in ergothioneine’s potential to modulate cellular aging.
This trial adds to the growing body of evidence supporting the favorable safety profile of ergothioneine. No adverse events attributable to ergothioneine were reported. Additionally, we observed potential hepatoprotective effects, with significant reductions in the plasma AST and ALT levels, particularly among males in the ERG 25 mg group. Preclinical findings concerning the hepatoprotective properties of ergothioneine have been reported, including its capacity to reduce oxidative stress, attenuate hepatic inflammation, and mitigate liver injury [68,69]. Importantly, plasma levels of TMAO, a metabolite implicated in cardiovascular risk, remained unchanged throughout this study, further supporting the safety of ergothioneine supplementation.
While this study has several strengths, including its randomized, double-blind design, high adherence to the study protocol, and inclusion of ITT and PP analyses to strengthen the reliability of the observed effects, this study also has a number of limitations. First, it should be noted that our original clinical trial registration included both the CNS-VS and COMPASS as primary outcomes, but through the development of our protocol and statistical plans, only CNS-VS Composite Memory was selected as the primary outcome in the finalized analytical plan to preserve statistical power. Despite this change, the observed objective effects were considered modest and the 16-week duration may not have been sufficient to detect between-group effects. The participants enrolled in our trial were relatively high functioning, with elevated and variable baseline ergothioneine levels, which may have led to ceiling effects. Additionally, this study was conducted during the COVID-19 pandemic, which may have influenced the study assessments and outcomes. Future studies should consider longer follow-up periods and consider stratification by baseline ergothioneine status or cognitive function to better characterize the efficacy.

5. Conclusions

This randomized, placebo-controlled trial provides preliminary insights into the cognitive and sleep-related effects of ergothioneine supplementation in healthy older adults with subjective memory complaints. While the primary outcome, composite memory, showed early improvement in the 25 mg group compared to baseline, this effect was not sustained and did not differ from placebo. Reaction time showed a significant treatment-by-time interaction favoring ergothioneine, yet the between-group differences were not significant, suggesting that any potential benefits were modest and require validation in larger or longer studies. Other cognitive effects observed were primarily within-group and not consistently dose-responsive, highlighting the challenge of detecting objective cognitive changes over a relatively short study duration in high-functioning healthy populations. However, positive effects of ergothioneine supplementation were observed on subjective measures of prospective memory and sleep initiation that were not seen in the placebo group. Importantly, ergothioneine supplementation was safe and well-tolerated, with no adverse events, favorable trends in liver function, and no increase in TMAO levels. Although median baseline ergothioneine levels were higher than the ‘healthy’ threshold proposed by Halliwell et al. [22], measurable beneficial effects were still observed, supporting the potential relevance of supplementation in healthy individuals with adequate ergothioneine status for supporting cognitive function. Overall, the findings of this trial should be considered largely preliminary and exploratory. Future studies with longer durations, as well as stratification by baseline ergothioneine or cognitive function, will be critical to fully determine the cognitive benefits of ergothioneine supplementation in aging.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/nutraceuticals5030015/s1, Table S1: Inclusion/exclusion criteria. Table S2: Description of CNS-VS domains. Table S3: Baseline descriptives for ergothioneine, telomere length, inflammation and liver function. Table S4: Baseline descriptives for cognitive function (CNS-VS), sleep quality (LSEQ) and subjective memory (PRMQ). Table S5: Within-group comparisons of the change (Δ) from baseline to 16 weeks in the CNS-VS domains. Table S6: Baseline descriptives for blood screening outcomes. Table S7: Blood biochemistry and treatment effects for ITT and PP. Figure S1. Leukocyte telomere length. Figure S2. Liver function (plasma AST and ALT). Figure S3. Plasma trimethylamine oxide levels.

Author Contributions

Conceptualization, I.T.Z., N.K. and L.S.M.-Z.; methodology, I.T.Z. and N.K.; software, B.K.-S.; formal analysis, B.K.-S.; investigation, I.T.Z. and N.K.; resources, I.T.Z., N.K. and L.S.M.-Z.; data curation, I.T.Z. and N.K.; writing—original draft preparation, I.T.Z., N.K., B.K.-S. and L.S.M.-Z.; writing—review and editing, I.T.Z., NK, B.K.-S. and L.S.M.-Z.; supervision, I.T.Z. and NK; project administration, I.T.Z. and N.K.; funding acquisition, L.S.M.-Z. All authors have read and agreed to the published version of the manuscript.

Funding

This trial was funded by Phyto Tech Corp (d/b/a Blue California).

Institutional Review Board Statement

This study was conducted in accordance with the principles of the Declaration of Helsinki. The study was approved by the CSIRO Human Research Ethics Committee (2021_015_HREC) and registered in the Australian and New Zealand Clinical Trials registry (ACTRN12621001607864).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Material. The raw data supporting the conclusions of this article can be made available on request. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

We acknowledge Steven Chen, Casey Lippmeier and Katie Ferren for their initial discussions related to the project conception and design, and Priscilla Samuel for her initiation of the clinical study at CSIRO and helpful input in the project design. We thank CSIRO’s clinical trial unit for executing the study, particularly Julia Weaver for her role as a clinical trial coordinator as well as Dana Pascovici for generating and formatting the figures. We thank SAPRO Consulting (Kim Steel and Kylie Tarascio) for serving as the local sponsor, with responsibilities including clinical trial registration, monitoring and documentation. We also thank all of the clinical trial participants.

Conflicts of Interest

Ian T. Zajac, Naomi Kakoschke, and Barbara Kuhn-Sherlock do not report any conflicts of interest, besides receiving funding for this study. Linda S. May-Zhang is employed by Blue California.

Abbreviations

The following abbreviations are used in this manuscript:
DASH Dietary Approaches to Stop Hypertension
MINDMediterranean–DASH Intervention for Neurodegenerative Delay
MCIMild Cognitive Impairment
CNS-VSCentral Nervous System Vital Signs
CSIRO Commonwealth Scientific and Industrial Research Organization
COMPASSComposite Autonomic Symptom Score
BMIBody Mass Index
MAC-QMemory Assessment Clinics Questionnaire
BPBlood Pressure
ERGErgothioneine
IWRSInteractive Web-Based Randomization Service
AHTAAdelaide Health Technology Assessment
GRASGenerally Recognized As Safe
cGMPCurrent Good Manufacturing Practices
PRMQProspective and Retrospective Memory Questionnaire
LSEQLeeds Sleep Evaluation Questionnaire
CRPC-Reactive Protein
EDTAEthylenediaminetetraacetic Acid
LC-QTofLiquid Chromatography Quadrupole Time-of-Flight
TMAOTrimethylamine N-Oxide
ELISAEnzyme-Linked Immunosorbent Assay
ITTIntention To Treat
PP Per Protocol
ANOVAAnalysis of Variance
MAC-QMemory Assessment Clinics Questionnaire
HSDHonestly Significant Difference
FDRFalse Discovery Rate
LSMLeast Squares Mean
CI Confidence Intervals
IQRInterquartile Range
MOCAMontreal Cognitive Assessment
GDSGlobal Deterioration Scale
SBPSystolic Blood Pressure
DBPDiastolic Blood Pressure
NCI Neurocognitive Index
ASTAspartate Aminotransferase
ALTAlanine Aminotransferase
TMA Trimethylamine

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Figure 1. CONSORT flow diagram of participant progression throughout the study.
Figure 1. CONSORT flow diagram of participant progression throughout the study.
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Figure 2. Plasma ergothioneine levels over time. Dose- and time-dependent increases in plasma ergothioneine levels were observed following supplementation with 10 mg and 25 mg ergothioneine compared to placebo (*** p < 0.001 vs. placebo). Values represent least-squares means with 95% confidence intervals from the intention-to-treat population.
Figure 2. Plasma ergothioneine levels over time. Dose- and time-dependent increases in plasma ergothioneine levels were observed following supplementation with 10 mg and 25 mg ergothioneine compared to placebo (*** p < 0.001 vs. placebo). Values represent least-squares means with 95% confidence intervals from the intention-to-treat population.
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Figure 3. Cognitive Function (CNS-VS) scores measuring changes from baseline. The standardized scores showed (A) a significant within-group improvement for Composite Memory (p < 0.05) for 25 mg ergothioneine at week 4 in the ITT sample, (B) a time-by-treatment interaction for Reaction Time (p < 0.05) in the ITT sample, and (C) an overall treatment effect for Psychomotor Speed (p < 0.05) in the PP sample. Values represent least-squares means with 95% Tukey-adjusted confidence intervals. Symbols: % = within-group effect; # = time-by-treatment interaction; $ = overall treatment effect.
Figure 3. Cognitive Function (CNS-VS) scores measuring changes from baseline. The standardized scores showed (A) a significant within-group improvement for Composite Memory (p < 0.05) for 25 mg ergothioneine at week 4 in the ITT sample, (B) a time-by-treatment interaction for Reaction Time (p < 0.05) in the ITT sample, and (C) an overall treatment effect for Psychomotor Speed (p < 0.05) in the PP sample. Values represent least-squares means with 95% Tukey-adjusted confidence intervals. Symbols: % = within-group effect; # = time-by-treatment interaction; $ = overall treatment effect.
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Figure 4. Subjective Prospective Memory. Average change from baseline in the Prospective Memory scores across the time points were plotted for each treatment group. A significant difference was observed in the ERG 25 mg group (p < 0.05 versus placebo), with a trend toward a dose-dependent effect in the ERG 10 mg group. Data shown are from the PP sample and represent least-squares means with 95% Tukey-adjusted confidence intervals. Symbols: * = effect compared to placebo.
Figure 4. Subjective Prospective Memory. Average change from baseline in the Prospective Memory scores across the time points were plotted for each treatment group. A significant difference was observed in the ERG 25 mg group (p < 0.05 versus placebo), with a trend toward a dose-dependent effect in the ERG 10 mg group. Data shown are from the PP sample and represent least-squares means with 95% Tukey-adjusted confidence intervals. Symbols: * = effect compared to placebo.
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Figure 5. Sleep initiation. Average change from baseline in the LSEQ “Getting to Sleep” scores across the time points. A significant overall treatment effect was observed (p < 0.05), driven by improvements in the ERG 25 mg group, with a trend toward a dose-dependent effect in the ERG 10 mg group. Data shown are from the PP sample and represent least-squares means with 95% Tukey-adjusted confidence intervals. Symbols: $ = overall treatment effect.
Figure 5. Sleep initiation. Average change from baseline in the LSEQ “Getting to Sleep” scores across the time points. A significant overall treatment effect was observed (p < 0.05), driven by improvements in the ERG 25 mg group, with a trend toward a dose-dependent effect in the ERG 10 mg group. Data shown are from the PP sample and represent least-squares means with 95% Tukey-adjusted confidence intervals. Symbols: $ = overall treatment effect.
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Table 1. Participant characteristics at determination of study eligibility.
Table 1. Participant characteristics at determination of study eligibility.
Placebo (N = 48)ERG 10 mg (N = 49)ERG 25 mg (N = 50)
CharacteristicN%N%N%
Female3572.93673.53672
Male1327.11326.51428
MedianIQRMedianIQRMedianIQR
Age (years)70.46.768.47.4697.9
MOCA272283282
MAC-Q272272271
GDS121212
SBP130.217.5132.718125.515.7
DBP78.713.378.797812
HbA1c (%)5.60.35.60.45.50.3
HbA1c (mmol/mol)374375374
Height (cm)163.514.3163.49.7165.312.5
Weight (kg)7416.973.41374.518.6
Body mass index27.75.227.65.526.15.3
Note: p-value for sex = chi-square; Kruskal–Wallis for other variables; IQR = interquartile range. MOCA = Montreal Cognitive Assessment; GDS = Global Deterioration Scale; SBP = systolic blood pressure; DBP = diastolic blood pressure.
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MDPI and ACS Style

Zajac, I.T.; Kakoschke, N.; Kuhn-Sherlock, B.; May-Zhang, L.S. The Effect of Ergothioneine Supplementation on Cognitive Function, Memory, and Sleep in Older Adults with Subjective Memory Complaints: A Randomized Placebo-Controlled Trial. Nutraceuticals 2025, 5, 15. https://doi.org/10.3390/nutraceuticals5030015

AMA Style

Zajac IT, Kakoschke N, Kuhn-Sherlock B, May-Zhang LS. The Effect of Ergothioneine Supplementation on Cognitive Function, Memory, and Sleep in Older Adults with Subjective Memory Complaints: A Randomized Placebo-Controlled Trial. Nutraceuticals. 2025; 5(3):15. https://doi.org/10.3390/nutraceuticals5030015

Chicago/Turabian Style

Zajac, Ian T., Naomi Kakoschke, Barbara Kuhn-Sherlock, and Linda S. May-Zhang. 2025. "The Effect of Ergothioneine Supplementation on Cognitive Function, Memory, and Sleep in Older Adults with Subjective Memory Complaints: A Randomized Placebo-Controlled Trial" Nutraceuticals 5, no. 3: 15. https://doi.org/10.3390/nutraceuticals5030015

APA Style

Zajac, I. T., Kakoschke, N., Kuhn-Sherlock, B., & May-Zhang, L. S. (2025). The Effect of Ergothioneine Supplementation on Cognitive Function, Memory, and Sleep in Older Adults with Subjective Memory Complaints: A Randomized Placebo-Controlled Trial. Nutraceuticals, 5(3), 15. https://doi.org/10.3390/nutraceuticals5030015

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