Transl Clin Pharmacol.

2023 Mar;31(1):13-27
https://doi.org/10.12793/tcp.2023.31.e1
pISSN 2289-0882·eISSN 2383-5427

Original Article Efficacy and safety assessment of

protein supplement - micronutrient
fortification in promoting health and
wellbeing in healthy adults -
a randomized placebo-controlled trial

Pranit Ambulkar 1,*, Prashant Hande 2, Bhagwat Tambe 3,

Vidyadhar G. Vaidya 4, Ninad Naik 4, Ramshyam Agarwal 5, and
Gayatri Ganu 6
1
R & D Healthcare, Netsurf Communications (P) Ltd., Laxman Nagar, Baner, Pune, Maharashtra 411045, India
Received: Oct 19, 2022 2
Analytical Research, Netsurf Communications (P) Ltd., Laxman Nagar, Baner, Pune, Maharashtra 411045, India
Revised: Dec 19, 2022 3
Food Technology, Netsurf Communications (P) Ltd., Laxman Nagar, Baner, Pune, Maharashtra 411045, India
Accepted: Dec 25, 2022 4
Lokmanya Medical Research Centre, Lokmanya Hospital, Chinchwad, Pune 411033, India
Published online: Mar 2, 2023 5
Health Nexus, Mangal Corner, next to Vedant Hospital, Thermax Chowk, Chinchwad 411018, India
6
Mprex Healthcare Pvt. Ltd. Wakad, Pune 411057, India
*Correspondence to
Pranit Ambulkar
Senior Manager R and D Healthcare, Netsurf
Communications (P) Ltd., Survey No. 107, Plot ABSTRACT
No. 2, Baner Rd, Laxman Nagar, Baner, Pune,
Maharashtra 411045, India.
Protein supplements are extensively used for muscle building, weight loss, recovery from
Email: Pranit.ambulkar@gmail.com
exercise, improving endurance & cardio-performance. Major challenge with protein
Copyright © 2023 Translational and Clinical supplement is undigested protein and impaired gut health which results in nausea,
Pharmacology dehydration, diarrhea, constipation, indigestion, stomach pain, and decreased appetite.
It is identical to the Creative Commons
Several studies have linked plant protein with reduced metabolic syndrome incidence.
Attribution Non-Commercial License (https://
Probiotics can improve gut health as well. The objective of the study is to assess the efficacy
creativecommons.org/licenses/by-nc/4.0/).
and safety of protein supplement in promoting health and wellbeing in healthy adults. The
ORCID iDs present trial is a double blind, multi-center, randomized, placebo controlled, clinical trial
Pranit Ambulkar involving 60 healthy individuals. The treatment duration was of 90 days. The subjects were
https://orcid.org/0000-0001-7699-2737
randomized to receive either protein supplement treatment or placebo control. Protein
Prashant Hande
https://orcid.org/0000-0002-0528-9363 supplement significantly improved quality-of-life score by 85.76%, VO2 max by 42.92%,
Bhagwat Tambe distance covered in 6 minutes, 100% individuals with at least 25% reduction in low energy
https://orcid.org/0000-0003-0132-3971 events as compared to the control group. Protein supplement treatment reduced body
Vidyadhar G. Vaidya weight (1.94 kg), waist circumference (2.46 cm), body mass index, waist circumference,
https://orcid.org/0009-0009-0249-2380
hip circumference and body fat. Remarkable and significant improvement in digestive and
Ninad Naik
https://orcid.org/0000-0003-2352-9479
sleep quality score, percent skeletal muscle was observed among protein supplement treated
Ramshyam Agarwal group. There were no clinically significant changes in hematological, biochemical and vital
https://orcid.org/0009-0004-8668-1325 parameters; indicating safety of protein supplement. Present study concluded that protein
Gayatri Ganu supplement is safe and efficacious in weight management, improving high energy events,
https://orcid.org/0000-0001-7812-3738 aerobic capacity, quality of life, digestive behavior score and sleep quality. This study ensures
Funding consumers about safety and effectiveness of protein supplement.
Supported by Netsurf Communication (P) Ltd.,
Pune, India for material, laboratory testing and Keywords: Protein; Probiotic; Endurance; Oxygen Consumption
other expenses of the trial.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Conflict of Interest INTRODUCTION

- Authors: Dr. Pranit Ambulkar, Mr. Prashant
Hande, Mr. Bhagwat Tambe are employee of
Protein and amino acid supplements are extensively marketed as muscle building and
the Netsurf Communication (P) Ltd.
- Reviewers: Nothing to declare
improving physical endurance in large consumer base like from athletes, active lifestyle
- Editors: Nothing to declare individuals to individuals with sedentary lifestyle [1-3]. In another way, low carbohydrate,
high protein diet has been used routinely as weight management regimen. Adults appears
Reviewer to be use protein supplement widely for health and fitness purposes like weight loss,
This article was reviewed by peer experts who
recovery from exercise, improving cardio-performance. In case of elderly individuals,
are not TCP editors.
largely the protein supplementation is intended at preserving the muscle mass, recovery
Author Contributions and maintaining well-being. Protein supplements in growing age are evident to provide the
Conceptualization: Ambulkar PY; Data favorable body conditions for growth, development and immunity [2]. There are various
curation: Ganu G; Formal analysis: Ganu
applications of a good protein source in keeping health at every age. The effectiveness of
G; Funding acquisition: Ambulkar PY;
Investigation: Vaidya VG, Naik N, Agarwal R; protein supplementation varies based on confounders like individual activity level, diet and
Methodology: Ganu G; Resources: Hande metabolic state [4,5].
P, Tambe B; Supervision: Vaidya VG, Naik
N, Agarwal R; Validation: Ambulkar PY; There are some challenges with high-protein diets that more undigested protein-derived
Visualization: Ambulkar PY; Writing - original
constituents end up in the large intestine leading to bacterial amino acid metabolism
draft: Ganu G; Writing - review & editing:
Ambulkar PY.
in the colon. The colonic fermentation of dietary amino acids may result in detrimental
effects to colonic health. From the previous research it is also evident that the protein
powder supplement consumers suffered from gastrointestinal symptoms, such as nausea,
dehydration, diarrhea, constipation, indigestion, stomach pain, and decreased appetite
[5,6]. On the contrary, plant proteins are found in plant foods, and positive health benefits of
plant foods are linked to dietary fiber, vitamins, minerals, and phytochemicals coming along
with the plant derived protein. Consumption of plant-based proteins are also correlated with
reduced incidence of metabolic syndrome [7]. It is well known fact that the gut microbiota
is an important factor for both energy harvest, storage and production through the protein
metabolism [8]. In order to improve the efficacy of protein supplementation and gut health,
probiotics either in protein supplement or together consumed is a proper strategy. Probiotics
can encourage host digestive protease and peptidase activity, and release exo-enzymes
involved in the protein digestion. Probiotics can improve the absorption of small peptides
and amino acids by improving the absorption ability of the epithelium and enhancing
transport [1]. The aim of the present study is to establish evidence of the efficacy and safety
of protein supplement along with probiotics, amino acid, vitamins, minerals and natural
ingredient rich fortification in promoting health and wellbeing in healthy adults.

Rationale of the study

The purpose of the research is evaluating safety and efficacy of protein supplement for
improving health and wellbeing in healthy individuals. There are many research articles
already proposing the health benefits of the key ingredients (plant-based proteins, milk
solids, micronutrients like vitamins, minerals, probiotics etc.) individually. It is hypothesized
that, protein supplement may provide health benefits to improve quality of life, energy,
endurance and digestive behavior compared to placebo (Milk solids).

Objectives of the study

The objectives of the study were to evaluate the efficacy, safety and tolerability of test product-
nutritional protein supplement in the improving health and wellbeing of healthy individuals.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

METHODS
Study design
We conducted a double blind, multi-center (2 study sites), randomized, placebo controlled,
clinical trial involving healthy individuals recruited from the outpatient department of
both the study centers i.e. Lokmanya Medical Research Centre & Hospital, Pune, India
and Health Nexus, Pune, India. The study was approved by Institutional Ethics Committee
(IEC) Lokmanya Medical Research Centre, Pune, India and Royal Pune Independent Ethics
Committee, Pune, India. The trial was registered on CTRI website (CTRI/2022/01/039219).
The trial involved parallel design, two groups i.e. protein supplement (Treatment) and
Placebo (control). 1:1 allocation of subjects was followed. The consolidated standards of
reporting trials (CONSORT) flow of the entire study are depicted in Fig. 1.

Inclusion criteria
Adults of age group 25–45 years old visiting outpatient department of study site were
considered for the study. Those willing to give written informed consent and, able to comply
with protocol requirements as per investigator judgement were selected. The criteria of
selection of subjects with body mass index (BMI) in the range of 20-26 kg/m2 with good
physical condition and sound mental status, willingness to abstain from use of vitamin or
mineral supplements, nutritional supplements and/or medical foods if applicable.

Screening

Assessment of eligibility (n = 68)

Excluded (n = 3)
- Not meeting inclusion criteria (n = 0)
- Declined to participate (n = 3)
- Other reasons (n = 0)

Randomized (n = 65)

Allocation

Allocated to protein supplement group (n = 32) Allocated to placebo group (n = 33)

Received allocated intervention (n = 32) Received allocated intervention (n = 33)

Follow-up

Lost to follow-up (n = 1) Lost to follow-up (n = 4)

(Due to the relocation) (Due to the relocation and non-feasibility to visit
to study centers due to personal reasons)

Analysis (n = 60)

Analyzed (n = 31) Analyzed (n = 29)

Figure 1. CONSORT diagram for the study.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Exclusion criteria
The subjects allergic to ingredients in product/ milk with uncontrolled and complicated
diabetes (HbA1C not more than [NMT] 5.7% and blood pressure NMT 140/90 mmHg) were
excluded from the study. Subjects with past history of addiction abuse, rehabilitation and more
than 2 events of alcohol consumption per week were not considered in the study. Subjects with
current medical history of any major illness such as cancer, heart disease, chronic obstructive
pulmonary disease, asthma etc. as well as subjects having any past history of any acute or
chronic illness that may affect the patient’s participation in the study were not considered for
participation. Subjects with acute illness or history of major or minor surgery in the past one
year, using prescription medications and/or nonprescription medications for weight loss, with
deranged liver function test (LFT) and kidney function test (KFT) as per investigator discretion
were not included in the study. Female subjects who are currently pregnant and/or breast
feeding were excluded. Volunteers not willing to participate in study were excluded.

Study groups
We intended to complete 60 subjects at the end of the study. We screened 68 participants
on above mentioned research sites based on the inclusion-exclusion criteria, of which three
withdrawn their consent before randomization and were considered screen failure. Total of 65
subjects entered the randomization. Participants were randomized using software i.e. SPSS
Version 10.0 (SPSS, Chicago, IL, USA) generated randomization sheet to receive either protein
supplement– treatment group and placebo – control group. Fig. 1 presents the flow of events for
the subjects considered in the analysis for this manuscript. We received randomization schedule
from qualified statistician, investigator enrolled the participants to respective study groups.

Sample size
We intended to enroll 60 subjects at the end of the study. We have considered comparison of
Mean Changes in total General Health Questionnaire-28 (GHQ-28) score between treatment
and placebo in 90 days (superiority study). 60 patients are required to have a 90% chance of
detecting, as significant at the 5% level, an increase in the primary outcome (reduction in
GHQ score) measure from 5% in the control group to 35% in the experimental group. Based
on this assumption from clinical experience, a qualified statistician evaluated the sample
size of total 60 (30 cases in each arm) completed cases needed to assess the study objective at
90% power and alpha 0.05. However, in the present study, the total evaluable subjects were
60 (31 in protein supplement– treatment group and 29 in placebo control group).

Intervention and dosage

Subjects from both the groups will be provided with container each containing 600 g of
protein supplement or placebo. Subject will be advised to consume given supplement in a
dose of 20 g in 200 ml milk once a day for 90 days.

Proposed intervention is a protein supplement. It is a proprietary product of Netsurf

Communication (P) Ltd, Pune, India. The product is composed of plant-based proteins, milk
solids, micronutrients like vitamins, minerals, probiotics like Bifidobacterium, Lactobacillus
together with some specialized natural ingredients like Colostrum, Omega 3 DHA, Aloe Vera
and Zeaxanthin etc.

Outcome measures
As protein supplement is composed of plant-based proteins, micronutrients, probiotics with
some specialized natural ingredients is thought to be provide health benefits to improve

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

quality of life, energy, endurance and digestive behavior in healthy individuals. The primary
outcomes were changes in total GHQ-28 score, serum total protein, and albumin to globulin
ratio, VO2 max by 6-minute walk test, BMI, fat %, skeletal muscle %, through impedance
studies for body composition along with waist and hip circumferences.

The secondary outcomes were change in % responders (subjects showing reduction of 25%
of low energy scores in 30 days’ time) on daily energy audit questionnaire using patient
diary. Energy audit questionnaire allows subject to record his/her experience, activity. As this
trial is a general representation of consumers of protein supplement, the subject reported
outcome on maintaining energy level could be an important parameter of determining
quality of life. This questionnaire was filled by subject every day in four different time
ranges i.e. morning, afternoon, evening and night time. Interpretation of energy was based
on total number of events from the category of high, moderate and low energy events. To
determine changes in total digestive behavior score all patients filled out a questionnaire
which included questions about the severity of GI symptoms during the last 4 weeks, rated
0–6, where 0 meant “no complaints” and 6 represented the worst imaginable severity of
that symptom. Severity score for all symptoms was analyzed as total mean score. Total sleep
quality score was evaluated using questionnaire- disturbed sleep cycle index. Patients were
asked to respond to questions and appropriate score was given (0-Did not apply to me at
all; 1-Applied to me to some degree, or some of the time; 2-Applied to me to a considerable
degree or a good part of time; 3-Applied to me very much or most of the time). Summing
up the score, sleep quality was assessed as mild (0-6), moderate (6-12), and severe (12-18).
Compliance of intervention was assessed. Safety was assessed by incidence of AEs and SAEs
either self-reported by subject or assessed by Investigator. There were no changes in the
outcome assessment and amendment to protocol.

Methodology
After receiving Ethics committee’s approval for study protocol, clinical study was registered
on CTRI website. subjects between 25 to 45 years of age attending outpatient/consultation
department of study site(s) will be screened for eligibility criteria.

On screening visit, a written informed consent was obtained from subject confirming
participation in the study. Subject’s demographic details were noted. Subjects underwent
clinical examination, complete blood count, LFT, KFT, HbA1c assessment. Subject’s medical,
surgical and treatment history was noted. VO2 max by 6-minute walk test and anthropometric
analysis was performed and documented. Subject’s current medication (s) if any were noted
in the case record from (CRF). Vitals were recorded. The subjects were considered for further
evaluation as per the inclusion and exclusion criteria.

During screening visit and the entire study duration subjects were advised to refrain from
any protein supplement, antioxidant agents, vitamins, anti-inflammatory drugs, hormones,
Nutraceutical, Ayurvedic, Siddha, Unani, herbal /homeopathic medicines for maintenance of
health. A screening window of 2-3 days was kept, in case if there is requirement of any tests
reports for confirming participation.

Subjects meeting all the inclusion criteria were randomized to the respective study groups
as per the computer-generated randomization list. Subject were asked for occurrence of
any adverse event during screening period. Subjects from the treatment group and placebo
group received respective medication for a total of 90 days. At randomization visit and at

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

every follow up visit (except last follow up visit), subjects were provided with container each
containing 600 g of protein supplement or placebo. Subjects were advised to consume given
supplement in a dose of 20 g in 200 mL milk once a day for 90 days. Subjects from placebo
group received identical investigational product like protein supplement. The packaging,
concealment method and flavor of the placebo were identical to that of the counterpart.

The record of concomitant medication other than prohibited ones, was kept in the CRF.
Subject were advised to continue the diet and exercise regimen (which they are already
following) during the entire study period of 90 days.

Investigational product compliance was assessed by the investigator on each and every follow
up visit. If subject continuously missed dosing for > 6 consecutive days or total missed dose
> 6 during the one assessment duration (one month), subject was treated as drop out of the
study. Subject was allowed to come for follow up either 1-2 days prior or after the scheduled
follow up visit, provided subject should continue the given treatment.

Subject were called at respective study sites for follow up visits after each 30 days for 90 days.
On baseline and every follow up visit, subjects underwent clinical examination. Subject were
assessed with some questionnaires related to health and wellbeing. On last follow up visit
(end of treatment visit) subject underwent clinical examination, complete blood count, LFT,
KFT, HbA1c assessment. VO2 max by 6-minute walk test and anthropometric assessment.
Subject were assessed with some questionnaires related to health and wellbeing.

After completion of 3 months of study treatment, subjects were asked to stop investigational
product and take advice of investigator for further treatment. Palatability with dosage
compliance was assessed from baseline to end of the study. Subjects were closely monitored
for any adverse event starting from baseline visit till the end of the study visit.

Data analysis
Patients without any major protocol violation, consumed at least one dose of intervention,
and those who did not take any prohibited medications during the study period were
considered for analysis. Continuous variables, such as age and other demographical
characteristics, were summarized by using summary statistics, i.e. frequency, and mean, and
standard deviation. Categorical values like gender and clinical examination were summarized
using frequencies and percentages.

Analysis of primary efficacy parameters

GHQ-28 score was analyzed by Student’s t-test, Mann Whitney U test. Changes in VO2 max by
6-minute walk test was represented using percentage and distance in meters. Other efficacy
parameters like serum total protein, albumin to globulin ratio, anthropometric parameters
were analyzed by Student’s- t test.

Secondary efficacy parameters

Changes in % responders (subjects showing reduction of 25% of low energy scores in 30
days’ time) on daily energy questionnaire was analyzed using Student’s- t test. Changes in
mean total digestive behavior score and total sleep quality score was analyzed by Student’s- t
test, Wilcoxon signed ranked test within groups and Mann Whitney U test between groups.
The adverse events were represented as number of events and analyzed by Fisher exact test.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

RESULTS
Total of 68 subjects were screened, of which three withdrawn their consent before
randomization and were considered screen failure. Total of 65 subjects entered the
randomization and were randomized in two groups (32 in treatment group and 33 in placebo
group). One subject from protein supplement group and four from placebo control group
were dropped out. Thus, we evaluated 31 subjects from protein supplement group and 29
from placebo control group (total of 60) in this manuscript.

Demographic characteristics
Both groups were comparable in terms of the mean age and gender distribution. The male to
female ratio in both test and control groups was comparable (Table 1).

Primary study outcomes

Change in total GHQ-28 score
All the patients were made to answer for the set of questions in the validated version of
GHQ-28. The GHQ-28 provides information on extent of psychosomatic symptoms which
can provide a fair understanding of overall quality of life. The four domains were considered
like somatic symptoms, anxiety & insomnia, social dysfunction, and depressive symptoms.
Self-scoring was done ranging from 0-1-2-3 (lower to higher disability respectively). The mean
score of all the questions and 4 sub-sections along with total GHQ score were calculated
and assessed. There is significantly more improvement in general health and wellbeing of
subjects in all four domains from treated group compared to placebo. Reduced score from
baseline indicated better quality of life. There was 85.76% improvement in total quality-of-life
score in treatment group compared to 28.47%in placebo group (Table 2)

Changes in mean serum total protein, and albumin to globulin ratio

There were no statistically (at p < 0.05) as well as clinically significant changes in albumin,
globulin, A/G ratio in both treatment and placebo groups (Table 3).

Changes in mean calculated VO2 max by 6-minute walk test

The aerobic capacity of the subjects was assessed by 6-minute walk test and VO2 max by
calculation was used as a tool to depict the improvement in the total oxygen consumption
capacity of individual. It was evident from the study that the baseline aerobic capacity was
comparable between groups, there was significant improvement in distance covered in
6 minutes by subjects in treatment group than placebo (Fig. 2). Consequently, there was
significant improvement in VO2 max of treatment group (42.92%) than placebo (6.77%)
depicted in Table 4.

Changes in anthropometric parameters

There was comparable body weight and BMI profile of subjects from both groups at baseline.
In treatment group, body weight was reduced with mean difference of 1.94 kg (p < 0.001),

Table 1. Demographic details of study subjects

Parameters Treatment Placebo
Group/Gender* Male (n = 20) Female (n = 11) Male (n = 22) Female (n = 7)
Age (yr)† 32.15 ± 7.11 31.36 ± 6.59 33.95 ± 6.18 29.43 ± 6.16
Total age (yr) 32 ± 6.9 33 ± 6.4
*Data analyzed by χ2 test. Not significant. p > 0.05.
†
Data analyzed by student t test.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Changes in distance (meter) covered in 6 minutes

800
Baseline Day 90
701.94
700

600
544.48
517.74 513.79
500

400

300

200

100

0
Treatment Placebo
Figure 2. Changes in distance covered in 6-minute walk test between groups.

Table 2. Changes in GHQ scores between groups

Duration Test Placebo p-value
Somatic symptoms score* (mean ± SD)
Baseline 13.80 ± 1.06 14.0 ± 0.90 0.4179
Day 90 1.90 ± 1.40 9.72 ± 1.47 < 0.001
% difference 86.23 30.57
Anxiety & insomnia score† (mean ± SD)
Baseline 13.40 ± 1.63 13.20 ± 1.28 0.5186
Day 90 2.51 ± 1.36 10.70 ± 10.70 < 0.001
% difference 81.27 18.94
Social dysfunction score* (mean ± SD)
Baseline 13.29 ± 1.41 13.40 ± 0.98 0.6312
Day 90 2.40 ± 1.50 10.93 ± 0.75 < 0.001
% difference 81.94 18.43
Depressive symptoms score† (mean ± SD)
Baseline 13.71 ± 1.41 13.10 ± 1.88 0.1618
Day 90 0.90 ± 0.74 6.90 ± 0.31 < 0.001
% difference 93.43 47.33
Total GHQ-28 score† (mean ± SD)
Baseline 54.16 ± 2.86 53.17 ± 2.13 0.1373
Day 90 7.71 ± 3.93 38.03 ± 2.03 < 0.001
% difference 85.76 28.47
GHQ-28, General Health Questionnaire-28.
*By Mann Whitney U test between groups. Significant at p < 0.05.
†
Data analyzed by Student’s t-test.

Table 3. Changes in liver function parameters between groups

Parameters Treatment group Placebo group
Baseline Day 90 Baseline Day 90
Bilirubin total 0.97 ± 0.33 0.88 ± 0.17 1.05 ± 0.41 0.92 ± 0.18
Bilirubin direct 0.34 ± 0.25 0.28 ± 0.10 0.37 ± 0.23 0.28 ± 0.11
Bilirubin indirect 0.63 ± 0.19 0.60 ± 0.12 0.68 ± 0.25 0.65 ± 0.12
SGOT 40.97 ± 23.47 33.68 ± 12.15 41.52 ± 28.00 32.03 ± 8.60
SGPT 33.87 ± 20.59 32.52 ± 17.82 34.49 ± 23.57 32.66 ± 12.18
Alkaline phosphatase 81.81 ± 22.93 84.42 ± 25.64 77.83 ± 17.72 84.59 ± 21.57
Total proteins 7.53 ± 3.11 7.11 ± 0.54 7.29 ± 0.80 7.09 ± 0.51
Serum albumin (A) 4.24 ± 0.44 4.06 ± 0.41 4.13 ± 0.39 4.03 ± 0.41
Serum globulin (G) 3.36 ± 1.67 3.04 ± 0.26 3.16 ± 0.51 3.08 ± 0.26
Serum A/G ratio 1.35 ± 0.61 1.31 ± 0.17 1.31 ± 0.18 1.28 ± 0.15
Data analyzed by Student’s t-test. Not significant at p < 0.05.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Table 4. Changes in Vo2 max between groups

Duration Mean ± SD (mL/kg/min)
Treatment Placebo p-value
Baseline 26.91 ± 4.33 26.56 ± 3.90 0.7454
Day 90 38.46 ± 4.38 28.36 ± 3.75
Mean diff (Baseline – Day 90) −11.55 ± 2.02 −1.80 ± 1.90 < 0.001
% Improvement 42.92 6.77
p-value 0.00002 0.00002
Data analyzed by Student’s t-test. Non-significant. p > 0.05.

BMI was significantly reduced by 2.86% (p < 0.001), waist circumference by 3.05% (2.46
cm reduction) (p < 0.001), hip circumference by 0.53% (p < 0.001) from baseline to end
of the study while there were no statistically significant changes in placebo-treated group.
Significant reduction in percent body fat by 2.46% (p < 0.001) in test group while, 0.85%
increase in placebo group was observed by end of the study. 1.57% (p < 0.01) of percent
skeletal muscle mass was increased in protein supplement treated group and there was no
significant change in placebo treated group (Table 5).

Secondary study outcomes

Changes in energy events (patient diary) score between groups
The energy audit was performed by calculating frequencies of low, moderate and high
energy events reported by subjects in their diaries for each 30 days till 90 days. It was evident
from data that there was significant improvement in high energy events in treatment group

Table 5. Changes in anthropometric parameters between groups

Duration Test Placebo p-value
Body weight (kg) (mean ± SD)
Baseline 64.89 ± 8.64 66.12 ± 8.29 0.5760
Day 90 62.96 ± 8.64 66.31 ± 8.17 < 0.001
% difference 2.97 0.28
p-value < 0.001 0.3656
BMI (kg/m2) (mean ± SD)
Baseline 23.01 ± 0.02 23.18 ± 2.17 0.7601
Day 90 22.35 ± 2.31 23.24 ± 2.16 < 0.001
% difference 2.86 0.25
p-value < 0.001 0.4108
Waist circumference (cm) (mean ± SD)
Baseline 80.43 ± 10.75 82.09 ± 9.39 0.5292
Day 90 77.97 ± 10.80 81.14 ± 9.29 0.00006
% difference 3.05 1.15
p-value < 0.001 0.0107
Hip circumference (cm) (mean ± SD)
Baseline 86.42 ± 9.98 89.98 ± 7.51 0.1259
Day 90 85.96 ± 9.96 89.59 ± 7.05 0.8484
% difference 0.53 0.43
p-value < 0.001 0.3060
Body fat (%) (mean ± SD)
Baseline 20.38 ± 3.89 19.86 ± 4.53 0.6398
Day 90 19.88 ± 3.52 20.03 ± 3.54 0.0421
% difference 2.45 0.85
p-value < 0.001 0.5476
Skeletal muscle (%) (mean ± SD)
Baseline 43.13 ± 5.45 42.70 ± 5.60 0.7630
Day 90 43.81 ± 5.66 42.43 ± 5.50 0.0076
% difference 1.57 0.63
p-value 0.0062 0.2846
Analyzed using Student’s t-test. Significant at p < 0.05.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Table 6. Changes in patient diary score between groups

Duration Test Placebo p-value
No. of high energy events (mean ± SD)
Day 30 0.29 ± 0.94 0.00 ± 0.00 0.1010
Day 60 12.16 ± 1.42 0.00 ± 0.00 < 0.001
Day 90 55.58 ± 3.91 17.28 ± 2.31 < 0.001
No. of moderate energy events (mean ± SD)
Day 30 111.06 ± 9.01 69.21 ± 14.62 < 0.001
Day 60 102.65 ± 6.19 71.21 ± 4.80 < 0.001
Day 90 57.00 ± 4.31 45.00 ± 4.73 < 0.001
No. of low energy events (Mean ± SD)
Day 30 8.65 ± 9.06 50.79 ± 14.62 < 0.001
Day 60 5.19 ± 5.86 48.79 ± 4.80 < 0.001
Day 90 7.42 ± 1.61 57.72 ± 4.22 < 0.001
Data analyzed by Student’s t-test. Significant at p < 0.05.

compared to placebo (p < 0.001). There were significantly more events in moderate to high
levels in treatment group at day 30, and the same trend followed till day 90 (Table 6).

To understand the response to improvement in energy levels we considered energy audit

score comparison between day 30 and day 60 between groups. There were 100% responders
i.e., individuals with at least 25% reduction in low energy events in treatment group whereas
there were only four (13.33%) responders in placebo group.

Assessment of symptom scores between groups

Digestive behavior score in treatment group was remarkably improved by 78.69%, 90.19%
100% at day 30, day 60 and day 90 from the baseline of the study (p < 0.001) in treatment
group while there was not significant change among placebo group. The sleep quality score
in treatment group was increased gradually and significantly (p < 0.001) by 31.54%, 32.69%
and 33.98% at day 30, day 60, day 90 respectively from baseline. No significant change was
observed in placebo group (Table 7).

Table 7. Changes in symptom scores between groups

Duration Test (% difference) Placebo (% difference) p-value
Digestive Behavior Score* (mean ± SD)
Baseline 0.61 ± 0.99 0.48 ± 0.91 0.7039
Day 30 0.13 ± 0.43 (78.69) 0.52 ± 0.78 (8.33)
Day 60 0.06 ± 0.25 (90.19) 0.48 ± 0.83 (0)
Day 90 0.00 ± 0.00 (100) 0.41 ± 0.63 (14.58)
Mean diff (Baseline – Day 30) 0.48 ± 1.00 −0.03 ± 0.82 0.0854
Mean diff (Baseline – Day 60) 0.55 ± 0.96 0.00 ± 0.60 0.0643
Mean diff (Baseline – Day 90) 0.61 ± 0.99 0.07 ± 0.88 0.0818
p-value 0.00007 0.9673
Sleep Quality Score† (mean ± SD)
Baseline 7.77 ± 1.67 7.31 ± 1.00 0.2007
Day 30 5.32 ± 1.28 (31.54) 6.76 ± 0.95 (7.52)
Day 60 5.23 ± 0.72 (32.69) 7.10 ± 0.67 (2.87)
Day 90 5.13 ± 0.72 (33.98) 7.07 ± 0.88 (3.28)
Mean diff (Baseline – Day 30) 2.45 ± 1.75 0.55 ± 0.83 < 0.001
Mean diff (Baseline – Day 60) 2.55 ± 1.61 0.21 ± 1.24 < 0.001
Mean diff (Baseline – Day 90) 2.65 ± 1.85 0.24 ± 1.46 < 0.001
p-value < 0.001 0.1318
*Data analyzed by Wilcoxan signed ranked test within groups and Mann Whitney U test between groups.
Significant at p < 0.05.
†
Data analyzed by Student’s t-test.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Table 8. Changes in hematological parameters between groups

Laboratory investigations Treatment Placebo
Baseline Day 90 Baseline Day 90
Total leukocyte count 8,145.16 ± 2,212.67 7,603.23 ± 1,762.48 8,493.10 ± 2,289.09 9,041.38 ± 3,392.69
Neutrophil % 58.87 ± 5.89 64.45 ± 8.19 59.31 ± 8.69 64.79 ± 8.91
Lymphocyte % 34.10 ± 5.56 29.32 ± 7.85 32.45 ± 7.42 28.79 ± 7.94
Monocyte % 3.42 ± 1.20 3.39 ± 1.58 3.83 ± 1.47 3.31 ± 1.61
Eosinophil % 3.61 ± 1.23 4.06 ± 1.26 4.41 ± 1.88 4.10 ± 1.57
Total RBC 4.63 ± 0.62 4.57 ± 0.48 4.90 ± 0.54 4.64 ± 0.56
Hemoglobin 12.90 ± 2.00 12.96 ± 1.77 13.78 ± 1.74 13.68 ± 1.56
Platelet count 228,451.61 ± 60,378.16 264,225.81 ± 85,040.27 208,379.31 ± 70,893.99 245,206.90 ± 69,788.14
Data analyzed by Student’s t-test. Not significant at p < 0.05.

Safety outcomes
There are no clinically as well as statistically (at p < 0.05) significant changes in
hematological, biochemical, and vital parameters in both groups post treatment (Tables 3, 8,
9, 10, and 11).

Adverse events
There were total 12 adverse events in the study duration, seven events in treatment group and
five in placebo group. The difference is not statistically significant. The adverse events were
headache, cough and cold, muscle pain, body ache, menstrual pain etc. The adverse events
were not related to the investigational product and were mild in nature.

There were 3 adverse events like bloating and hyperacidity in treatment group compared to 7
adverse events related to gastro intestinal tract in placebo group. These adverse events were
possibly related to investigational product but were mild in nature and required no change in
dosage and got relieved in 1–2 days without rescue medication.

Table 9. Changes in kidney function test parameters between groups

Laboratory Investigation Treatment Placebo
Baseline Day 90 Baseline Day 90
Serum calcium 9.79 ± 3.10 9.85 ± 0.73 9.85 ± 2.71 9.78 ± 0.80
Serum uric acid 4.76 ± 1.67 4.50 ± 1.25 5.05 ± 1.43 4.58 ± 1.14
Blood urea nitrogen 12.42 ± 3.05 14.05 ± 3.90 12.13 ± 4.26 13.68 ± 3.35
Serum creatinine 0.96 ± 0.18 0.98 ± 0.17 0.99 ± 0.25 1.03 ± 0.16
BUN/creatinine ratio 13.10 ± 3.07 14.19 ± 4.53 12.39 ± 4.51 13.08 ± 2.94
Data analyzed by Student’s t-test. Not significant at p < 0.05.

Table 10. Changes in HbA1c parameters between groups

Laboratory investigation Treatment Placebo
Baseline Day 90 Baseline Day 90
HbA1c 5.53 ± 0.51 5.56 ± 0.44 5.70 ± 0.43 5.62 ± 0.47
Data analyzed by Student’s t-test. Not significant at p < 0.05.

Table 11. Changes in vitals between groups

Laboratory investigation Treatment Placebo
Baseline Day 90 Baseline Day 90
Pulse 72.52 ± 1.06 72.23 ± 1.36 72.24 ± 1.30 71.62 ± 1.95
Temperature 97.72 ± 0.77 97.63 ± 0.80 97.71 ± 0.75 97.68 ± 0.77
Systolic BP 121.52 ± 3.53 120.90 ± 2.49 121.38 ± 3.23 121.28 ± 3.26
Diastolic BP 81.23 ± 1.75 80.74 ± 2.00 80.93 ± 2.07 81.14 ± 1.64
Respiratory rate 16.16 ± 1.00 16.52 ± 1.03 16.55 ± 1.02 16.24 ± 0.95
Data analyzed by Student’s t-test. Not significant at p < 0.05.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Compliance
There was high i.e. 92% and 90% compliance observed in protein supplement and placebo
group, respectively. There were total 5 drop outs from the study population, 1 from treatment
group and 4 from the placebo group respectively. Reasons for drop-out from the study were
relocation and non-feasibility to visit to study centers due to personal reasons and not the
compliance.

DISCUSSION
In the present study, we compared the impact of protein supplement with micronutrient
fortification versus placebo in promoting health and wellbeing in healthy adults. Baseline
status of the participants in both groups were comparable. All the subjects were representing
sedentary or slightly active lifestyle. This avoids the bias and promised data integrity and
robustness. After treatment with protein supplement, subject’s general health and wellbeing
was significantly improved. 85.56% improvement in quality of life was observed. No change
was observed in albumin, globulin and A/G ratio by the end of the study in both the groups.
Aerobic capacity was increased in individuals in protein supplement treated group which was
calculated by 6-minute walk test (VO2 max). Significant reduction in BMI, waist circumference,
% body fat and increased % skeletal muscle mass in treatment group was observed as compared
to placebo group. There was remarkable improvement in number of high energy events in the
protein supplement group participants compared to placebo group. There was improvement
in digestive behavior and sleep quality in protein supplement group than placebo group. There
were no clinically significant changes in hematological, biochemical and vital parameters were
observed in both groups indicating safety of investigational product.

Gastrointestinal discomfort especially due to milk based marketed products, weight gain,
unhealthy spike in blood sugar, dehydration, diarrhea, nausea is some of the common side
effects associated with use of protein supplement. A systematic review of experimental
and randomized studies analysis revealed that chronic, and excessive use of protein
supplementation may cause adverse events related to kidney and liver function, impaired
bone metabolism, and increased low density lipoprotein (LDL) [9,10]. All proteins are
not easily digested and have varied absorption due to either their source or processing
conditions. In recent years there is great interest amongst researchers to study various
benefits of plant-based proteins for improving human health. Plant protein are linked
with benefits for health and physical function. Plant proteins in the diet can help to supply
adequate high-quality protein and also reduces the potential for adverse impact [11,12]. There
is increased consumer interest in protein consumption, as well as increased interest in plant-
based diet patterns. In the present research, protein supplement possess proteins obtained
from rice, pea and soya, together with milk and whey proteins.

Probiotic microorganisms mainly used in human nutrition are species of Lactobacillus and
Bifidobacterium. The beneficial effects of probiotics can be used to restore the natural microbiota,
counter attack pathogenic intestinal microbiota activity and in digestive processes, the
management of food allergies, candidiasis, and tooth decay. Probiotics increase the efficiency
of the immune system, absorption of vitamins and minerals, and also stimulate the production
of amino acids [13]. As it is evident from the results, probiotics used in the formulation that is
Bifido longum and Lactobacillus acidophilus could have potentially reduced gastrointestinal problems
evident by negligible side effects of the investigational protein supplement.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Recommendation for daily intake of protein is 0.83 g protein/kg/day in healthy adults

regardless of sex [14]. Protein intake is vital to encourage healthy weight loss, treat
protein deficiency, help build muscle, aid muscle recovery, perform integral functions like
immunological response and to promote general wellbeing of individuals. In athletes and
sportspersons protein is needed to enhance endurance and promote muscle growth [1,15,16].

The previous research suggests that, adequate dietary protein supplementation is important
for overall health and improving body composition. When protein and energy intakes unable
to meet individual nutritional demand, body stores are catabolized to provide energy, leading
to the depletion of muscle mass with resulting symptoms such as fatigue or tiredness.
Poor nutritional status is also associated with depleted energy levels and reduced physical
performance leading to fatigue [17].

As per present study, protein supplement treated group reported better quality of life,
improvement in general health and wellbeing, improved energy levels throughout the day.
The effectiveness of the protein supplement can be attributed to the synergistic effect of
combination of multiple plant proteins, milk protein, probiotics and various micronutrients
within their standardized specifications.

Reduced muscle mass, strength, and cardiorespiratory fitness, resulting in an impaired

ability to perform daily activities could be the signs of biological aging. Improved maximal
oxygen uptake (VO2 max) is a hallmark of physical endurance [18]. In this present study, we
have studied effects of protein supplement treatment on VO2 max without any resistance or
physical training protocol. There is significant improvement in VO2 max by 42.92% in protein
supplement treated group compared to their baselines. It can be assuring parameter for
overall improvement in health and activity levels.

Treatment with protein supplement led to reduction in BMI, waist circumference and %
body fat which makes it potential candidate for weight management which is very crucial
parameter for metabolic health.

Protein supplement can be considered as safe presenting no clinical, vital, hematological

and biochemical signs of any abnormal changes including very few, minor in nature adverse
events which are not related to study intervention. Product is well tolerable and safe for long
term (90 days) use.

The strength of the study is it provides a real time evidence of combination of plant
and animal-source based protein and its effectiveness in the healthy population. Study
participants were individuals without comorbidities and major health concerns. There is
significant improvement in energy levels, general health wellbeing, digestion of formulation,
sleep quality, aerobic capacity, quality of life and anthropometric parameters. Present study
is more generalizable, relatable to consumers. The fewer sample size could be thought as the
limitation of the study. Further research can be carried out with protein supplement using
different age groups, gender, groups with varied activity levels and additional comorbidities.

It can be concluded from the present study that protein supplement is safe and efficacious
in promoting general health and wellbeing in adults. It can help in weight management with
some more dietary modifications, as it is observed to reduce BMI, hip circumference, waist
circumference, % body fat. It ensures improvement high energy events, aerobic capacity.

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Clinical study to evaluate safety and efficacy of protein supplement in healthy adults

Treatment with protein supplement ascertained the fact that it can provide a wholesome
improvement in quality of life, digestive behavior score and also improves sleep quality. There
was improvement in percent skeletal muscle measured by impedance study.

Negligible gastrointestinal side effects of protein supplement are more assimilated and
provided the sustainable results in promoting health and wellbeing. The present research
ensures consumers of this product to get the effectiveness to remain energetic, healthy and
manage weight.

ACKNOWLEDGMENTS
The authors would like to acknowledge the research team and the back-office team involved
in the research work. We would like to acknowledge the support provided by back office,
Netsurf Communication (P) Ltd.

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