This study evaluates whether a combination of plant protein with adaptogenic herbs (ashwagandha and rhodiola) can improve sleep quality in middle-aged adults with sleep disturbances.

One hundred participants aged 35-70 years with sleep problems will be randomly assigned to receive either the active intervention or placebo for 30 days. The active group will receive 27 grams per day of plant protein (pea, rice, and hemp isolate) combined with 300 mg of Rhodiola rosea and 500 mg of Withania somnifera (ashwagandha KSM-66®) in tablet form. The placebo group will receive maltodextrin and placebo capsules that look identical.

Sleep quality will be measured daily using a validated Sleep Quality Questionnaire (SQQ) that assesses difficulty falling asleep, daytime sleepiness, and overall sleep quality. Body composition and cellular health will also be measured using bioimpedance analysis at the beginning and end of the study.

The primary hypothesis is that the combination of plant protein with ashwagandha and rhodiola will produce greater improvements in sleep quality compared to placebo. The study will also evaluate the safety and tolerability of this nutritional intervention.

BACKGROUND AND RATIONALE

Sleep disturbances affect 30-40% of the adult population worldwide and are associated with serious health consequences including insulin resistance, weight gain, and increased cardiovascular risk. While pharmaceutical sleep medications (benzodiazepines and hypnotics) are commonly prescribed, they have significant limitations including potential for addiction, adverse side effects, and disruption of natural sleep architecture.

Natural alternatives are increasingly sought. Protein supplementation may influence sleep through multiple mechanisms: proteins provide tryptophan (a precursor to serotonin and melatonin), help stabilize nocturnal blood glucose levels, and can generate bioactive peptides with sedative properties. However, studies on protein and sleep have shown mixed results.

Adaptogens are medicinal plants that help the body adapt to stress. Ashwagandha (Withania somnifera) has been extensively studied and shown to improve sleep quality, reduce sleep latency, and decrease nighttime awakenings, likely through modulation of the hypothalamic-pituitary-adrenal (HPA) axis and GABAergic effects. Rhodiola rosea, another well-characterized adaptogen, has demonstrated stress-resilience enhancement and neuroprotective effects.

This study is the first to investigate the combined effects of plant protein with both ashwagandha and rhodiola on sleep quality, hypothesizing a synergistic multimodal effect.

STUDY DESIGN

This is a randomized, double-blind, placebo-controlled, parallel-group clinical trial conducted at three sites in Spain (Pozoblanco, Seville, and Yecla). The study was approved by the Research Ethics Committee of the Virgen Macarena and Virgen del Rocío University Hospitals (Code: 2061-N-21) on June 21, 2021, and is conducted in accordance with the Declaration of Helsinki.

PARTICIPANTS

One hundred adults aged 35-70 years with sleep disturbances will be recruited and randomized 1:1 to intervention (VERUM, n=50) or placebo (PLACEBO, n=50) groups. Key inclusion criteria include experiencing sleep disturbances, willingness to consume the plant protein supplement, and ability to provide informed consent. Exclusion criteria include known allergies to study ingredients, current use of sleep medications, serious medical conditions, and pregnancy or lactation.

INTERVENTION

VERUM Group: Participants will receive plant protein powder (27 g/day containing pea, rice, and hemp protein isolate) plus two tablets daily containing Rhodiola rosea (300 mg) and Withania somnifera KSM-66® (500 mg) for 30 consecutive days.

PLACEBO Group: Participants will receive maltodextrin powder (matched for appearance and taste) plus placebo capsules (identical in appearance) for 30 consecutive days.

All participants and investigators will be blinded to group assignment throughout the study period.

OUTCOME MEASURES

Primary Outcome: Sleep quality will be assessed using the validated Sleep Quality Questionnaire (SQQ), completed daily by participants. The SQQ evaluates three domains:

• Sleep Difficulty subscale (range 0-16)
• Daytime Sleepiness subscale (range 0-24)
• Total Sleep Score (range 0-40)

Higher scores indicate worse sleep quality.

Secondary Outcome: Phase angle, a bioelectrical parameter reflecting cellular health and membrane integrity, will be measured using bioimpedance analysis (BIA-TELELAB) at baseline (Day 0) and end of intervention (Day 30).

STATISTICAL ANALYSIS

Sleep quality data will be analyzed using longitudinal mixed models to account for repeated daily measurements. Phase angle will be analyzed using non-parametric tests. Effect sizes will be calculated using Cohen's d. The significance level will be set at α = 0.05. Analysis will include all participants who complete outcome assessments.

PROPOSED MECHANISMS

The proposed mechanisms of action include: (1) tryptophan provision for serotonin/melatonin synthesis, (2) HPA axis modulation and cortisol reduction, (3) GABAergic effects, (4) nocturnal glycemic stabilization, and (5) enhanced stress resilience through adaptogenic pathways.

SAMPLE SIZE JUSTIFICATION

A sample size of 100 participants (50 per group) was determined to provide adequate statistical power to detect clinically meaningful differences in sleep quality measures between groups, accounting for an estimated dropout rate of 20-30%.

SAFETY MONITORING

Participants will be monitored for adverse events throughout the study. Any adverse events will be recorded and reported according to institutional and regulatory requirements.

Inclusion Criteria:

• Adults aged 35 to 70 years (inclusive)
• Experiencing sleep disturbances or sleep quality problems
• Willing and able to consume plant protein supplement daily for 30 days
• Willing and able to take adaptogen tablets daily for 30 days
• Able to provide written informed consent
• Able to complete daily sleep quality questionnaires
• Available for baseline and follow-up assessments
• Willing to maintain regular lifestyle habits during the study period

Exclusion Criteria:

• Age less than 35 years or greater than 70 years
• Known allergies or hypersensitivity to any study ingredients (pea protein, rice protein, hemp protein, Rhodiola rosea, Withania somnifera/ashwagandha, or excipients)
• Current use of prescription sleep medications or hypnotics
• Current use of other herbal supplements for sleep (washout period may be allowed)
• Serious or unstable medical conditions that could interfere with study participation or safety
• Severe psychiatric disorders requiring medication
• Uncontrolled metabolic diseases (for example, uncontrolled diabetes, thyroid disorders)
• Pregnancy or lactation
• Planning to become pregnant during the study period
• Participation in another clinical trial within the past 30 days
• Inability to understand or comply with study procedures
• Shift workers or individuals with irregular sleep schedules
• Recent major life events significantly affecting sleep patterns

Individual participant data that underlie the results reported in the published article will be available upon reasonable request to qualified researchers for meta-analyses or other legitimate research purposes. Data will be shared after de-identification to protect participant privacy.

Individual participant data (IPD) will be available beginning 6 months after publication of the primary study results and will remain available for 5 years following publication. Requests received after this period will be considered on a case-by-case basis depending on data storage and accessibility at that time.

IPD will be shared with qualified researchers who provide a methodologically sound proposal for legitimate research purposes, including meta-analyses, systematic reviews, or secondary analyses. Requestors must:

1. Submit a detailed research proposal describing the aims, methods, and planned analyses

2. Demonstrate appropriate ethical approval from their institution (if required)

3. Sign a data access agreement committing to:

   • Use data only for approved research purposes
   • Maintain participant confidentiality
   • Acknowledge the original study in any publications
   • Not attempt to re-identify participants

Requests should be directed to the corresponding author at fjberde@upo.es (Dr. Francisco José Berral-de la Rosa, Universidad Pablo de Olavide, Seville, Spain). Proposals will be reviewed within 30 days. Approved requestors will receive de-identified IPD in a secure format.

Participants were randomly assigned to one of two parallel groups (VERUM or PLACEBO) for the entire 30-day intervention period. No crossover or sequential assignment was used.