Plant Protein With Ashwagandha-Rhodiola for Sleep Quality

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.

• Plant Protein Supplement (Dietary supplement), also known as Pea Protein Isolate, Rice Protein Isolate, Hemp Protein Isolate: Plant-based protein powder supplement containing 27 grams per day of a proprietary blend of three protein sources: pea protein isolate, rice protein isolate, and hemp protein isolate. The powder is mixed with water or beverage of choice and consumed once daily for 30 consecutive days. This protein blend provides essential and branched-chain amino acids, including tryptophan (precursor to serotonin and melatonin), which may influence sleep quality through neurotransmitter synthesis and nocturnal glycemic stabilization.
• Rhodiola rosea Extract (Dietary supplement), also known as Rhodiola, Golden Root, Arctic Root: Rhodiola rosea extract supplement in tablet form containing 300 milligrams per day. One or two tablets taken daily with meals for 30 consecutive days. Rhodiola rosea is an adaptogenic herb that helps the body manage stress through modulation of the hypothalamic-pituitary-adrenal (HPA) axis. It enhances stress resilience and has neuroprotective effects. May synergistically improve sleep quality when combined with other adaptogens and protein supplementation.
• Ashwagandha KSM-66 (Dietary supplement), also known as Withania somnifera, KSM-66 Ashwagandha, Indian Ginseng: Withania somnifera (Ashwagandha) KSM-66® extract supplement in tablet form containing 500 milligrams per day. One or two tablets taken daily with meals for 30 consecutive days. Ashwagandha is an adaptogenic herb that modulates the hypothalamic-pituitary-adrenal (HPA) axis, reduces cortisol levels, and has gamma-aminobutyric acid (GABA) receptor effects. Extensively studied for improving sleep quality, reducing sleep latency, and decreasing nighttime awakenings.
• Placebo (Dietary supplement), also known as Placebo Comparator, Maltodextrin: Placebo intervention consisting of two components: (1) Maltodextrin powder matched to the active protein powder in appearance, taste, texture, color, and packaging, mixed with water or beverage, consumed once daily; and (2) Inert placebo capsules identical in appearance, size, color, shape, and packaging to the active adaptogen tablets, taken with meals. All placebo products contain no active ingredients and are consumed for 30 consecutive days. Participants, care providers, investigators, and outcomes assessors are blinded to treatment assignment.

Primary outcomes

• Sleep Quality assessed by Sleep Quality Questionnaire (SQQ) [Daily assessments throughout the 30-day intervention period (Day 1 to Day 30)]: Sleep quality will be measured using the validated Sleep Quality Questionnaire (SQQ), a self-reported instrument completed daily by participants. The SQQ evaluates three domains: 1. Sleep Difficulty subscale (range: 0-16 points) 2. Daytime Sleepiness subscale (range: 0-24 points) 3. Total Sleep Score (range: 0-40 points) Higher scores indicate worse sleep quality. Lower scores indicate better sleep quality. The questionnaire assesses difficulty falling asleep, nighttime awakenings, sleep quality perception, and daytime functioning related to sleep.

Secondary outcomes

• Phase Angle measured by Bioimpedance Analysis (BIA) [Baseline (Day 0) and End of Intervention (Day 30)]: Phase angle is a bioelectrical parameter that reflects cellular health and membrane integrity, measured using bioelectrical impedance analysis (BIA) with the BIA-TELELAB device. Phase angle is derived from the relationship between resistance and reactance of body tissues to an electrical current. It is expressed in degrees (°). Higher phase angle values generally indicate better cellular health, membrane integrity, and body cell mass. Lower values may indicate cellular dysfunction or compromised health status. The measurement will be performed under standardized conditions (fasting, hydration controlled, supine position).

Sex: All, ages: 35 Years to 70 Years

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

• Results publication: Molina-López A, Leo-Romero A, Molina-López M, Polo-Marco S, Domínguez-Balmaseda D, García-Pérez-de-Sevilla G, Casanova E, Moya-Amaya H, Granizo Bermejo D, Berral-de la Rosa FJ. Effects on Sleep Quality and Bioelectrical Parameters of Cellular Health After Administration of a Plant Protein Supplement Combined with Ashwagandha and Rhodiola: A Randomized, Double-Blind, Placebo-Controlled Trial. Manuscript in preparation. 2026.

• Francisco José Berral-de la Rosa, Principal investigator, PhD