The Effects of Ashwagandha (Withania Somnifera) Supplementation on Exercise Performance in Female Footballers
Phase: NA
Conditions: Muscle Strength
Sponsor: Blanca Roman-Viñas, MD

 
 
 
 
 
 
Title: The Effects of Ashwagandha (Withania Somnifera) 
Supplementation on Exercise Performance in Female Footballers 
 
Reference: NCT06264986 
 
Document date: 01/04/2024 (submitted 11/11/2024) 
 
 

 
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Table of Contents 
Title ......................................................................................................................................... 3 
Summary ................................................................................................................................. 3 
Background and current status ................................................................................................. 5 
Justification of the study .......................................................................................................... 6 
References ............................................................................................................................... 7 
Hypothesis ............................................................................................................................... 8 
Objectives ................................................................................................................................ 9 
Methodology ........................................................................................................................... 9 
Ethical considerations ............................................................................................................ 15 
Work plan .............................................................................................................................. 20 
Resources .............................................................................................................................. 21 
Applicability and limitations ................................................................................................... 22 
Dissemination plan ................................................................................................................ 23 
References ............................................................................................................................. 23 
Appendix ............................................................................................................................... 30 
Sample Size Calculation ................................................................................................................................30 
Borg Scale .....................................................................................................................................................32 
Total Quality Recovery questionnaire ...........................................................................................................33 
Hooper Index questionnaire .........................................................................................................................34 
Supplement satisfaction questionnaire ........................................................................................................35 
Agreement document from club ..................................................................................................................36 
 
 
 

 
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Title 
The effects of ashwagandha (Withania somnifera) supplementation on exercise performance 
in female footballers. 
 
Summary 
English: The objectives of the thesis are to determine the effects of ashwagandha (ASH) on 
muscle strength, muscle recovery and to assess habitual dietary intake of female footballers. 
Across one phase, two experimental research papers and one descriptive research paper will 
be produced; it will involve one double-blind, randomised and placebo-controlled trial and a 
comparative analysis of nutritional intake in female footballers. The thesis will involve 
participants supplementing ASH in the form of a capsule containing KSM-66 (600 mg with 
30mg withanolides) or placebo once a day for 28 days, on a singular occasion. The objectives 
of the first two papers are to determine the effects of ASH on muscle strength, perception of 
recovery, perception of exertion, perceived wellness and perceived muscle soreness. For the 
paper on muscle strength, the methodology will include hand grip test, counter movement 
jump (CMJ), squat jump (SJ), peak power, medicine ball throw and rate of perceived exertion 
(RPE) via the Borg Scale. For the paper on muscle recovery, the methodology will include a 
questionnaire on sleep, stress, fatigue and delayed onset of muscle soreness (DOMS) test via 
the Hooper Index (HI), total quality recovery (TQR) questionnaire and a supplement 
satisfaction questionnaire. The objectives of the third paper are to evaluate the dietary intake 
of professional female footballers and compare the values against published nutritional 
recommendations. The methodology will involve self-tracking of dietary intake via the Snap-
N-Send method and data will be stored and analysed with the software Nutritics. The study 
will be conducted with a professional club in Barcelona. The use of ASH is becoming prevalent 
in athletes, despite lack of information regarding dosage guidelines or lack of research on its 
benefits for exercise performance. The proposed study will contribute to the overall scientific 
knowledge of ASH and whether it may demonstrate benefits from short-term use. 
Additionally, the study will determine if there are benefits for female athletes that wish to 
increase strength and reduce perception of pain or soreness related to exercise. The ethical 
considerations for this study are predominantly the safety and comfort of the participant. An 
informed consent form will be given to the participant, containing information about data 
collection, personal information, safety of the supplement and their right to drop out of the 

 
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study at any point. The proposed start date is January 2024, and the study will collectively last 
for 36 days across a singular phase. The supplement is provided by Zenement España, a 
supplement company based in Barcelona. 
 
Spanish: Los objetivos de la tesis son determinar los efectos de la ashwagandha (ASH) sobre 
la fuerza muscular y la recuperación muscular, así como evaluar la ingesta dietética habitual 
de las futbolistas. Se elaborarán dos estudios, uno de investigación experimental y un segundo 
de investigación descriptiva; el primero implicará un ensayo doble ciego, aleatorizado y 
controlado con placebo y el segundo un análisis comparativo de la ingesta nutricional en 
futbolistas femeninas. El primer estudio requerirá que las participantes tomen suplementos 
de ASH en forma de una cápsula que contenga KSM-66 (600 mg con 30mg withanólidos) o (en 
el grupo control) un placebo, una vez al día durante 28 días, en una sola toma. Los objetivos 
del primer estudio son determinar los efectos del ASH sobre la fuerza muscular, la percepción 
de la recuperación, la percepción del bienestar, la percepción del esfuerzo y las agujetas. Se 
presentará un artículo con datos sobre la fuerza muscular, en el que la metodología incluirá 
pruebas de agarre con la mano, salto con contramovimiento (CMJ), salto en cuclillas (SJ), 
potencia máxima, lanzamiento de balón medicinal y el índice de esfuerzo percibido (RPE) a 
través de la escala de Borg. Un segundo artículo evaluará la recuperación muscular y el 
bienestar, la cual se medirá mediante una prueba de valoración del sueño, el estrés, la fatiga 
aparición retardada de dolor muscular (DOMS) mediante el Hooper Index (HI), un cuestionario 
de recuperación de calidad total (TQR) y un cuestionario de satisfacción de uso del 
suplemento. Los objetivos del segundo estudio (y tercer artículo) son evaluar la ingesta 
dietética de las futbolistas profesionales y comparar los valores con las recomendaciones 
nutricionales publicadas. La metodología consistirá en el auto-registro de la ingesta dietética 
a través del método Snap-N-Send; posteriormente los datos se almacenarán y analizarán con 
el software Nutritics. El estudio se realizará con un club profesional de Barcelona. El uso de 
ASH es cada vez más frecuente en los atletas, a pesar de la falta de información sobre las 
pautas de dosificación o la falta de investigación sobre sus beneficios para el rendimiento en 
el ejercicio. El estudio propuesto contribuirá al conocimiento científico general sobre el ASH; 
por una parte, pretende demostrar los beneficios de su uso a corto plazo, y por otra, el estudio 
determinará si existen beneficios para las atletas que desean aumentar su fuerza y reducir la 
percepción de dolor o molestias relacionadas con el ejercicio. Las consideraciones éticas para 

 
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este estudio son predominantemente la seguridad y la comodidad del participante. Se 
entregará a los participantes un formulario de consentimiento informado con información 
sobre la recogida de datos, la anonimidad de los datos personales, la seguridad del 
suplemento y su derecho a abandonar el estudio en cualquier momento. La fecha de inicio 
propuesta es enero de 2024, y el estudio durará colectivamente 36 días en una fase singular. 
El suplemento lo suministra Zenement España, una empresa de suplementos con sede en 
Barcelona. 
 
Background and current status 
Ashwagandha (ASH) is a medicinal plant with adaptogenic properties, clinically studied to 
demonstrate anti-stress, anti-inflammatory, anti-tumour, immunomodulatory, analgesic, 
neuroprotective and antioxidative effects (Bonilla et al., 2021). Those effects have encouraged 
intrigue for the use of ASH in sports performance. Notably, a study by (Wankhede et al., 2015) 
studied the effects of ASH on muscle strength and reported significant increases after 8 weeks 
of supplementation alongside a resistance training programme. The study has its limitations 
as participants’ food habits were not considered, and subjects were previously untrained and 
exclusively male. The experimental group demonstrated considerable physiological benefits 
post-intervention compared to the placebo group, but more controls are needed in future 
studies to affirm the effects of ASH and muscle strength. A 4-week study by (Tripathi et al., 
2016) explored muscle power alongside VO2 max among males. Improvements were found in 
speed, distance travelled, and muscle power (measured with a hand grip dynamometer) 
compared to the control group. Despite favourable results, limitations include being an open-
label study, exclusively male participants and no food diary of participants. A 12-week study 
by (Ziegenfuss et al., 2018) researched the effects of ASH on perceptive pain, body strength 
and a cycling time trial. Improvements were shown in upper and lower body strength, 
perception of recovery and muscle soreness. There are strengths to this research, such as 
keeping track dietary intake during the study, well-trained participants and a double-blind 
control. However, the limitations include an exclusively male sample and questionable cycling 
time trial results that do not reflect the scores of highly trained individuals. None of the 
participants in the above studies had any adverse reactions. Even with the aforementioned 
limitations, the above studies are heavily referenced as confirmation that ASH is a ‘proven’ 
beneficial supplement for exercise performance. More studies with better controls and 

 
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monitoring of confounding factors are needed to verify the efficacy of ASH in exercise 
performance. Additionally, new research should be performed on participants who are 
female, highly trained or athlete level and dietary intake should be closely monitored. 
 
Justification of the study 
The justification to study female footballers is due to the lack of research in this specific area. 
There are public calls within sports science to expand research on performance and explore 
methods of how players can improve performance and the recovery process to avoid 
disruption of play and training. A study by (O’Donnell et al., 2018) noted the connection 
between cortisol and sleep: a 354% increase in cortisol post-match was found in elite female 
athletes, resulting in significantly reduced sleep time, quality and quantity; these factors are 
crucial in not only the recovery process but to general wellbeing and performance. Some of 
the ASH benefits are explained by its role in cortisol regulation in the HPA axis. If ASH reduces 
raised cortisol in female athletes, there is evidence that it could lead to improved sleep and 
as a result benefit overall health and performance. The objective of the thesis is to determine 
whether four weeks supplementation of ASH has the ability to improve muscle strength 
performance and perception of wellness and muscle soreness. 
 
There is a fast-growing interest in ASH among practitioners, athletes and active individuals. 
However, there is an emerging amount of anecdotal feedback from users who supplement 
ASH in extreme doses and for a long period of time that they experience anhedonia or 
emotional numbness as a side effect. This is echoed in clinical research, where the active 
ingredient of ASH, called withanolides, is given to rats in extreme high doses (equivalent to 7-
20 commercial tablets daily) for a long duration; the side effects are likened to antidepressants 
such as lorazepam (Bhattacharya et al., 2000). This may be due to continuous buffering of 
cortisol in the HPA axis (Lopresti et al., 2019). Additionally, current published research on ASH 
and exercise is not of high quality, as voiced by Asker Jeukendrup, one of the world’s leading 
sports nutritionists (Jeukendrup, 2023). Current studies on ASH and exercise are mostly 
between 8-12 weeks. Despite no adverse reactions or toxicology reported in any of the clinical 
studies, in the author’s opinion, this time length of study runs the risk of an anhedonia 
response due to consistently reduced cortisol. There are no general guidelines on usage, but 
the sports industry is amplifying the use of ASH by accrediting products as Informed Sport 

 
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Certified (specifically KSM-66, a third-party tested, branded product of ASH that is extracted 
from the root and without the use of alcohol or chemical solvents). If one commercial tablet 
of ASH supplemented daily for four weeks shows physiological benefits, it is in the athlete’s 
best interest to cycle ASH intermittently instead of ingesting it continuously for a long 
duration. If four weeks of ASH supplementation does not exhibit effects, the conversation 
should begin on whether it is a beneficial supplement to use in general. 
 
The justification to monitor professional female footballers’ habitual food intake is to assess 
whether they receive adequate nutritional intake to complement their match and training 
schedule. It is opportunistic research, as food diaries will already be logged during the 
familiarisation of the muscle strength and recovery study. However, it is beneficial to analyse 
current dietary habits and compare it against recently published recommendations for female 
footballers (Dobrowolski et al., 2020). The benefit of this analysis is to potentially provide 
sports nutrition practitioners with insight on areas that may require improvement or attention 
in regard to female footballer’s dietary intake and how they can be further supported. 
 
References 
1. Bonilla DA, Moreno Y, Gho C, Petro JL, Odriozola-Martínez A, Kreider RB. Effects of 
Ashwagandha (Withania somnifera) on Physical Performance: Systematic Review and 
Bayesian Meta-Analysis. Journal of Functional Morphology and Kinesiology. 2021 Feb 
11;6(1):20. 
 
2. Wankhede S, Langade D, Joshi K, Sinha SR, Bhattacharyya S. Examining the effect of 
Withania somnifera supplementation on muscle strength and recovery: a randomized 
controlled trial. Journal of the International Society of Sports Nutrition [Internet]. 
2015;12(1):43. 
 
3. Tripathi R, Salve B, Petare A, Raut A, Rege N. Effect of Withania somnifera on physical 
and cardiovascular performance induced by physical stress in healthy human 
volunteers. International Journal of Basic & Clinical Pharmacology. 2016;5(6):2510–6 
 

 
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4. Ziegenfuss T, Kedia A, Sandrock J, Raub B, Kerksick C, Lopez H. Effects of an Aqueous 
Extract of Withania somnifera on Strength Training Adaptations and Recovery: The 
STAR Trial. Nutrients. 2018 Nov 20;10(11):1807. 
 
5. O’Donnell S, Bird S, Jacobson G, Driller M. Sleep and stress hormone responses to 
training and competition in elite female athletes. European Journal of Sport Science. 
2018 Feb 26;18(5):611–8. 
 
6. Bhattacharya SK, Bhattacharya A, Sairam K, Ghosal S. Anxiolytic-antidepressant activity 
of Withania somnifera glycowithanolides: an experimental study. Phytomedicine. 2000 
Dec;7(6):463–9. 
 
7. Lopresti AL, Smith SJ, Malvi H, Kodgule R. An investigation into the stress-relieving and 
pharmacological actions of an ashwagandha (Withania somnifera) extract: A 
randomized, double-blind, placebo-controlled study. Medicine. 2019;98(37):17186. 
 
8. Jeukendrup 
A. 
Is 
ashwaganda 
a 
performance 
supplement? 
[Internet]. 
askerjeukendrup. 2023. Available from: https://www.mysportscience.com/post/is-
ashwaganda-a-performance-supplement 
 
9. Dobrowolski H, Karczemna A, Włodarek D. Nutrition for Female Soccer Players—
Recommendations. Medicina. 2020 Jan 10;56(1):28. 
 
Hypothesis 
1. Short-term ASH has beneficial effects on muscle strength in female footballers 
2. Short-term ASH has positive effects on perception of muscle recovery following 
intense exercise in female footballers 
3. Female footballers are not consuming recommended levels of nutrients for optimal 
sports performance 

 
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Objectives 
1. Evaluate the effect of ASH on muscle strength in female footballers 
2. Evaluate the effect of ASH on the perception of recovery in female footballers 
3. Evaluate habitual dietary intake female footballers 
 
Methodology 
Design 
1. Muscle strength and recovery RCT: Double-blind, randomised, placebo-controlled trial. 
2. Food intake analysis: Observational, descriptive and cross-sectional research design 
 
Subjects 
The study population will be female footballers who belong to a professional club. Specifically 
female footballers who are signed to CE Seagull in Barcelona, Catalunya. 
 
Sample size 
The sample size was generated using G*Power (Version 3.1.9.6). Using the peak power 
variable in a study analysing ASH and muscle soreness (Diehl, 2021), the estimate of the effect 
size of Cohen’s d equals to 0.53 and therefore used in the sample size calculation for this 
proposed research. There is no further calculation to convert Cohen’s d into Cohen’s f to make 
it appropriate for ANOVA, as when there are only two groups Cohen’s f is the same as Cohen’s 
d (University of Michigan, 2021). Using ANOVA repeated measures between factors, effect size 
of 0.53, 80% power, 5% risk error in a two-parallel-group design and three measurements, the 
overall sample size is 30 participants, with 15 subjects per group. The calculation provided by 
G*Power can be found in the appendix. 
 
Selection criteria 
Inclusion for participants who are: 
• Female 
• Playing football professionally 
• At a sub-elite to elite standard 

 
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• Healthy and free of disease 
 
Exclusion for participants with: 
• Active supplementation with other ergogenic aids 
• Medication or contraceptives (to not interfere with existing treatment) 
• Thyroid conditions (due to interaction with cortisol) 
• Diabetes or certain autoimmune conditions (to not interfere with existing 
treatment) 
• Active pregnancy (to not interfere with existing treatment) 
• Allergies to nightshades such as tomato, aubergine, potatoes and peppers (as ASH 
is an herb in the nightshade group) 
• No signing of the consent form 
 
The withdrawal criteria will be participants who request to withdraw and do not wish to 
proceed, or do not complete the required study tests. 
 
Intervention 
The intervention will be 600mg of KSM-66, a branded supplement derived from the root 
extract of the herb ashwagandha, with a consistent rate of 5% withanolides for 28 days. It is 
the most clinically studied extract of the herb and is the only type of ashwagandha to receive 
third party testing certification (Informed Sport, Informed Ingredient, BSCG, Clean Label 
Project). The laboratory where the product is made, Ixoreal Biomed, has received Current 
Good Laboratory Practices (cGLP), quality controlled and tested against heavy metals and 
pesticides. 
 
A highly important factor of KSM-66 is its sole use of extraction from the root of ashwagandha, 
thereby avoiding the leaves. Extracts derived from the leaf contain Withaferin A and 
Withanone, which are considered cytotoxic (Priyandoko et al., 2011; Khedgikar et al., 2015; 
Rossato Viana et al., 2022). A paper by (Siddiqui et al., 2021) states that Withanone is the 
active metabolite that can induce liver toxicity with overuse. In a previous case report by 
(Björnsson et al., 2020), liver injury of moderate severity was reported in five patients after 

 
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supplementing ashwagandha. The listed ashwagandha supplements responsible for the 
injuries had no information on extraction process or what part of the herb was used, no 
certification and had unstated levels of withanolides. 
 
This amplifies the importance of sole root extraction, controlled levels of withanolides and 
third-party testing for ashwagandha supplements, such as KSM-66. The safety and tolerability 
of root-extracted KSM-66 is evident in scientific literature (Raut et al., 2012; Sharma et al., 
2018; Salve et al., 2019). In a trial by (Verma et al., 2021b) body weight, body temperature, 
pulse rate, respiratory rate, systolic and diastolic blood pressure, haematological and 
biochemical parameters were analysed to be normal in males and females after 8 weeks of 
daily consumption of 600mg KSM-66. 
 
Muscle strength will be predominantly based on the data from the hand grip test using the 
hand-held dynamometer, CAMRY model. The participants will squeeze the dynamometer with 
all their ability on each hand, and the strength values will be determined by an average of 
each hand’s measurement. 
 
During the 28 days, participants will have 12 training sessions and 4 official matches. It is likely 
the participants will experience menstruation during the study. However, recent findings 
suggest there is no change in strength or exercise performance from short-term fluctuations 
in reproductive hormones (Colenso-Semple et al., 2023). 
 
Dosage  
For 28 days, participants in the experimental group will ingest 600mg KSM-66 (30mg 
withanolides) once daily at midday with a meal. Participants in the control group will ingest a 
HPMC capsule containing 600mg of chickpea flour once daily at midday with a meal.  
 
Potential benefits 
Benefits will vary between subjects, however ASH supplementation may: 
• Reduce levels of cortisol, resulting in: 
o Improved sleep 
o Improved muscle strength 

 
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o Improved cardiorespiratory fitness 
o Improved perception of recovery 
o Reduced difficulty of exertion during exercise 
(Bonilla et al., 2021) 
Potential side effects 
Side effects of ASH are uncommon, however large doses (exceeding 1,250mg daily, based on 
a clinical trial examining tolerability) may induce: 
• Direct irritation to the intestinal mucosa, resulting in: 
o Nausea 
o Vomiting 
o Gastrointestinal upset 
(Raut et al., 2012, LiverTox, 2019) 
 
Regarding the potential side effects of KSM-66 specifically, a trial by (Chandrasekhar et al., 
2012) noted adverse effects in subjects after 60 days of 600mg KSM-66 supplementation. 
Overall, there were 6 adverse events with ASH and 5 with placebo. They included congestion, 
decreased appetite, cough and cold symptoms. The placebo group experienced fever, 
headache, tiredness and abdominal pain, making it unclear as to whether KSM-66 has notable 
or established side effects. 
 
Intervention group 
The intervention for the experimental group will be KSM-66 ashwagandha (600mg 
ashwagandha containing 30mg withanolides, once a day with meals for 28 days, ingested at 
midday) supplied by the company Zenement based in Barcelona. The branded product of 
KSM-66 is made by Ixoreal Biomed. 
 
Control group 
The control group will receive rice flour in a HPMC capsule, once a day with meals for 28 days, 
ingested at midday. The rice flour will be a commercial product found in supermarkets. To 
achieve randomisation, simple random allocation will be used to prevent bias amongst 
participants. The protocol will follow the randomisation guide by (Arifin, 2012) and will be 

 
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achieved in SPSS software. It will be a double-blind study as the main researcher will not know 
which participants supplement ASH or placebo. 
 
Variables 
Dependent: 
Muscle strength research: 
• Hand grip strength in kilograms (kg) using a CAMRY dynamometer. The variable of 
interest will be the mean of the right and the left-hand measurements 
• Seated medicine ball throw in metres (m) 
• Counter movement jump (CMJ) in watts per kilogram (W·kg-1) 
• Squat jump (SJ) in centimetres (cm),  
• Peak power in watts per kilogram (W·kg-1), using the results from SJ 
• Rate of Perceived Exertion (RPE) via Borg Scale 
 
Muscle recovery research: 
• Perceived ratings of sleep, stress, fatigue and Delayed Onset Muscle Soreness (DOMS) 
via Hooper Index (HI) 
• Total Quality Recovery (TQR) questionnaire 
• Supplement satisfaction questionnaire 
 
Food diary analysis research, compared against (Dobrowolski et al., 2020): 
• Energy intake (kcal) 
• Energy availability (kcal/kg fat free mass/day) 
• Protein (g/day) 
• Carbohydrates (g/day) 
• Fat (%) 
• Fluids (L/day) 
• Vitamin D (mg/day) 
• Calcium (mg/day) 
• Iron (mg/day) 
 

 
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All data will be collected via Snap-N-Send and stored using Nutritics dietary analysis software 
(Research Edition, v5.096, Dublin, Nutritics, 2019). The Snap-N-Send method was analysed in 
a doubly labelled water study and was shown to have enhanced validity for assessing athlete 
total energy intake (Costello et al., 2019). The Nutritics software was clinically studied to be a 
useful and valid tool for healthcare professionals to measure macronutrients and 
micronutrients, demonstrating that its analysis values positively correlated with plasma 
markers (Watkins et al., 2020) 
 
Independent: 
• The treatment of ASH: 600mg KSM66 (30mg withanolides) once a day for 28 days 
 
Data collection 
Study data will be collected manually and entered into Blanquerna’s OneDrive system. All data 
sheets and files will be backed up on to Google Sheets and shared with associated researchers 
and tutors of the project. This allows for data to be reviewed by collaborators. 
 
Dietary intake data will be collected via Whatsapp using Snap-N-Send. The data will then be 
manually entered and stored using Nutritics and access will be shared with collaborators. 
 
Data analysis 
For the muscle strength and recovery research, dependent variables will be described as the 
mean and standard deviation and calculated using and ANOVA repeated measures, between 
factors as there will be three time points. A descriptive analysis will be carried out for all the 
data collected in the data collection notebook. To assess the normality of the data distribution, 
the Kolmogorov-Smirnov test will be used. 
 
For the food diary analysis, dependent variables will be described as the mean and standard 
deviation and determined using one-sided t-test to compare against data averages of 
nutritional recommendations for female footballers (Dobrowolski et al., 2020). Pearson’s r will 
be used to examine correlations between the selected variables. 
 

 
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Ethical considerations 
The ethical considerations for this study are predominantly the safety, comfort and consent of 
the participant: 
• Epistemic injustice will be avoided in the study by the researcher listening to the 
participant and valuing their opinion and feelings on the research subject 
• The safety and tolerability of ASH has been clinically studied in humans and mice and 
is deemed safe to ingest. However, participants will be informed of rare, potential side 
effects and their right to leave the study at any time 
• Personal information (name, contact details) of the participant will not be shared with 
a wider network or in the research papers, but will be used by the researcher during 
the study to check-in on the participant. They will be contacted for their questionnaire 
scores during the research on perceptive muscle recovery – if the participant does not 
agree to this method, they may decline or drop out of the study 
• There will be data collection during the study, and it is important to not violate the 
participants right to privacy when asking personal questions via the questionnaires. 
The participant may decline to answer any question they deem unsuitable 
• Participants will be made aware that they will be observed for the data collection 
during the study and may decline this if it makes them uncomfortable 
• If the researcher fails to respect personal, cultural or religious values that makes the 
participant feel uncomfortable, they have the right to drop out of the study 
• The participants will be informed about the aims of the study and will be asked to sign 
an informed consent form (the form can be found in the appendix) 
 
Scientific and social value 
The study is contributing to the generation of scientific knowledge. For a supplement that is 
widely used, the existing knowledge on its effects on athletes and females is substantially low. 
In addition, there is very little existing research on short-term supplementation of ASH as the 
focus is currently on 8-12 weeks supplementation. This study will inform as to whether the 
supplement can provide performance benefits after 28 days, as the author believes this is a 
more realistic and safer dosage duration. This research therefore holds relevance in scientific 
advancements and may provide new dosage guidelines to users. Another aspect of scientific 

 
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relevance is the response to the public calls for more research on this specific area of ASH and 
sports performance. 
 
There is high social relevance and value for the female athlete population, who are likely to 
experience strict training schedules with limited recovery time between training and matches. 
If significant benefits are found in recovery from ASH, there is a safe and low-cost method for 
athletes to adapt into their routine. Additionally, if there are benefits found in this thesis, 
practitioners who work directly with female athletes will be informed of up-to-date practice 
with ASH and can pass on information to athletes. Importantly, if the thesis shows no benefits 
across any measures, both athletes and practitioners will be informed of the supplement and 
its credibility. 
 
The study holds high social and innovative relevance as it will collect, summarise and present 
existing published research that indicates a) overuse in high dosage and duration may lead to 
consistently lowered cortisol and b) consumption of solely root extraction of the herb is highly 
recommended in order to avoid potentially ingesting the cytotoxic metabolites of Withaferin 
A and Withanone, which are found in the leaves of the herb. Providing safety and information 
for users and future users of ASH is the crux of this thesis. 
 
Scientific-technical validity 
All of the instruments and questionnaires used in the proposed study are established in peer-
reviewed literature and deemed both valid and reliable. Two measures will be taken for each 
tool. The first instance will be the baseline measure and will occur on-site at the training 
ground gym in the evening during a typical training session. The second measure will take 
place after 28 days of supplementation (half ASH, half placebo) in the same location and time 
of day. Each participant will be given a timeslot to enter the gym and perform their 
assessments before continuing with normal resistance-training session. The tests using 
instruments will be monitored by two strength and conditioning coaches, and the data will be 
collected by two sports scientists. The following evaluation instruments are as follows: 
• Hand grip strength of the participants will be measured with a CAMRY dynamometer 
and the predominant measure to assess muscle strength. It is a commonly used tool 
in sports performance and demonstrates validity and reliability in relation to athlete 

 
17 
strength (Cronin et al., 2017). The validity for the use of a CAMRY dynamometer is 
documented by (Huang et al., 2022) when comparing against the Jamar dynamometer. 
The CAMRY dynamometer is inexpensive, easily accessible and provides valid results 
• Counter movement jump (CMJ) and squat jump (SJ) jumping tests will be used to 
assess estimation of muscle power in the lower limbs of the participants. These tests 
will be performed in the on-site gym at the football club, using appropriate equipment 
to carry out the tests. Valid and reliable scores are obtained via CMJ and SJ (Markovic 
et al., 2019) 
• Seated medicine ball throw will be used to assess explosive power in the upper body. 
Participants will sit on the floor with legs fully extended and back against the wall to 
throw a 4kg medicine ball as far as possible. This test has validity and reliability when 
assessing muscle strength and general athletic ability (Stockbrugger & Haennel, 2001) 
• Peak power will be calculated using the centimetres measurement of SJ with the 
following formula: (60.7) × (jump height [cm]) + 45.3 × (body mass [kg]) − 2055 (Sayers 
et al., 1999). The reliability and validity of average peak power is acceptable (Natera et 
al., 2023) 
• Participants will be asked to provide their rate of perceived exertion (RPE) after the 
previous tests are complete. This will be retrieved using the Borg Scale (Borg, 1998). It 
is a popular tool in published literature on sports performance, and provides a valid 
and reliable measure of exercise intensity and exertion from the participant (Lea et al., 
2022) 
• The following day, post-resistance training recovery will be assessed by the Total 
Quality Recovery (TQR) questionnaire, developed by (Kenta y Hassmén, 1998). This 
questionnaire monitors sleep quality, emotional state, stress and other subjective 
markers following exercise. Its validity is documented by (Osiecki et al., 2015), when 
compared against creatine kinase (CK) levels in football players; a marker that has been 
found to be a strong indicator of stress and recovery in athletes. The high CK levels 
were significantly associated with the lower the TQR score, making the subjective 
questionnaire relevant to physiological recovery. The TQR questionnaire is a valid 
alternative to evaluate recovery and is easy applied in this proposal 

 
18 
• On the following day, participants will be asked to rate their delayed onset of muscle 
soreness (DOMS), sleep, stress and fatigue via Hooper Index. The Hooper Index is a 
low-cost monitoring tool to measure wellness markers, such as perceived sleep quality, 
stress, fatigue and muscle soreness. It is shown to strongly correlate with exercise 
fatigue in professional soccer players (Rabbani et al., 2019) 
• The final evaluation tool will be a supplement satisfaction questionnaire. This will ask 
participants to rate their satisfaction with the supplement, either ASH or placebo, on 
a scale between 1 and 5 (1 = very satisfied; 5 = very dissatisfied). They will additionally 
be asked to measure their willingness to use it in future (1 = very unwilling; 5 = very 
willing). This questionnaire has been used by (Kuehl et al., 2010b) in a previous study 
evaluating muscle pain following a course of supplementation 
• The food diaries of participants will be received via Whatsapp by the Snap-N-Send 
method, which is a titled and photography-based mobile assessment and considered 
a valid and reliable method for assessing energy intake (Costello et al., 2017). 
• The tool to analyse nutritional data will be Nutritics, electronic software that is 
commonly used to analyse food diaries. Electronic diet assessments are scientifically 
valid to measure macronutrients and micronutrients, and there is additional benefit 
from being low-cost (Raatz et al., 2015; Ferrara et al., 2019). 
 
Equitable selection of subjects 
Recruitment and selection of participants will be aided by the selected liaison of the football 
club, who are already familiar with the club structure, the players and the personal health 
information of the players. Presentations will be given during training sessions about the 
study. During the presentations with the desired participant groups, all the information of the 
study will be displayed with easily understandable and digestible information, without stating 
misleading or false expectations. Published research about the supplement will be included 
in one of the presentation slides for transparency. In the presentation it will be made clear 
that participants may receive either the supplement or a placebo. It will be made clear that 
participation is optional and not mandatory. It will be made clear there is no reward or 
compensation for joining the study, except for receiving a food report back with nutritional 
guidance to optimise their sports performance (as participants will have been tracking their 

 
19 
diary throughout the study) provided by the author and the collaborator. As the author and 
in-house collaborator are registered sports nutritionists, the reports hold authenticity. After 
the presentation has finished, participants can independently choose to sign up. The 
presentation would be done a month before the study would begin, giving subjects time to 
consider their participation and to sign the consent forms. In the presentation the following 
selection criteria will be shown: 
 
Benefit / risk ratio 
There is minimal risk posed to participants involved in this study, as there are no reported 
harms from supplementing KSM-66 in clinical research and no adverse effects have been 
reported. Additionally, it is a commercially available product that has been certified safe to 
supplement. As KSM-66 is made from root extraction of the ashwagandha herb, and therefore 
not made of the leaves, there is less toxicological risk as the metabolites Withaferin A and 
Withanone are removed in the process (further elaboration in the Thesis – Intervention 
section of this document) making KSM-66 safer than other products of ashwagandha.  
Potentially, a participant could have an undiagnosed illness or allergy to nightshades and the 
supplement gives side-effects. Previous literature has not reported any instances of this 
happening. It is unlikely as athletes typically have a medical team at their disposal, who 
provide health checks and make sure they are fit to play. Due to the short-term time length 
and reasonable dosage of the study, there is minimal risk for over-supplementation leading to 
consistently reduced cortisol or gastrointestinal problems. There is low to no risk pertaining 
to psychological, social or economic factors. The study has been designed with participant 
comfort in mind and the timeline, training sessions, performance and recovery tests mimic 
the participants’ usual regime, allowing for ease of participation and reduced risk factor. 
 
The anticipated benefits outweigh the risks. The supplement of KSM-66 has shown to improve 
muscle strength, recovery, cognition and sleep in published literature. These are highly 
important factors to the lifestyle of an athlete and provide social, psychological and physical 
benefits.  
 
Privacy 

 
20 
All personal data and information collected or generated in the study will be stored on 
OneDrive Blanquerna and protected in accordance with the current legislation on personal 
data protection (Regulation (EU) 2016/679 and in the Organic Law 3/2018 of 5 December on 
Data Protection). No one, except some of the staff directly related to this study, will be able to 
know the identity. Data will be stored only for the duration of the study and during the process 
of analysing the data. 
 
Integrity 
There is no known conflict of interest in this study. There will be no plagiarism within this study 
and all and words will be original to the study. There will be no fabrication or invention of 
results, and data will be analysed and published accurately and transparently. All collaborators 
will be employed staff members of the football club who are collaborating in the thesis. The 
gym facilities where the tests will be measured and performed are on-site of the football club 
and where the players have weekly gym training sessions. The gym facility is a licensed 
establishment. The participants will be informed of the results and will have access to their 
individual data. 
 
Work plan 
A visual representation of the study timeline can be found in the appendix. A description of 
each step is as follows: 
0. Approval from the ethical committee 
1. Participants are informed about the aims of the study and sign the consent form. 
Information about age, BMI, years of practice and health history is collected 
2. Participants start tracking their food diaries via an app remotely for three days over 
the weekend prior to the supplementation for familiarisation 
3. Baseline measures for muscle strength will be taken on the following day at the club 
gym, coinciding with a regular gym training session. The measures will be taken prior 
to starting a typical training session 
4. The team will consist of the author (Olivia Coope), Andrea Páez Calvente and Andrea 
Reales Salguero who will assist with data collection and physiotherapist Enrique Jordán 
who will monitor participants and their safety while performing the strength tests 

 
21 
5. Afterwards participants will be given a printout of the TQR questionnaire and the 
Hooper Index to complete for the next day. Participants will be given their course of 
supplementation, either placebo or KSM-66, and instructed to take one a day for 28 
days 
6. The following day, baseline measures for the muscle recovery will be collected 
remotely at midday via Whatsapp or Zoom, and at the same time participants will start 
supplementation of either KSM-66 or placebo 
7. Fourteen days later, participants perform the muscle strength tests in the evening at 
the club gym. Similar to before, the team will consist of the author (Olivia Coope), 
Andrea Páez Calvente, who will assist with data collection and physiotherapist Enrique 
Jordán who will monitor participants’ safety while performing the strength tests 
8. Afterwards participants continue with the regular gym training session. Printouts of 
TQR and Hooper Index questionnaires are given to participants for the next day 
9. The following day muscle recovery measures are collected remotely via Whatsapp or 
Zoom at midday 
10. An additional fourteen days later, participants finish supplementation and perform the 
muscle strength tests in the gym with identical circumstances (staff, equipment, 
temperature) as the previous two gym sessions 
11. The following day muscle recovery measures are collected remotely via Whatsapp or 
Zoom at midday. The trial finishes 
12. The analysis of data begins 
13. The process dissemination of results and findings begins 
 
The study is designed with ease of participation in mind, but there will be inconveniences for 
participants who forget to take the intervention or fill out the food diary. To alleviate this, 
participants will be notified (with permission via the consent form) over Whatsapp with 
reminders during the 28 days supplementation. Additionally, there will be a check-in session 
a week after supplementation starts, to assess participant satisfaction and compliance. 
 
Resources  
Primary resources will consist of raw data through observation obtained from gym equipment 
and hand grip dynamometer and first-hand accounts from participants when answering 

 
22 
subjective questionnaires and food diaries. Secondary resources will consist of Excel and 
Google sheets to store the data, SPSS software to analyse the data, and Nutritics software to 
analyse the dietary intake. 
 
The facility where the baseline and post-supplementation muscle strength tests are 
performed will take place at the club gym for CE Seagull (located at Travessera de Montigalà, 
18, 08917 Badalona, Barcelona). Participants will be familiar with the facility and its 
equipment due to previous weekly gym training sessions prior to any measurements. 
 
Applicability and limitations 
Applicability 
There is a high likelihood that the study may impact the target population of female footballers 
and to athletes as a wider population. The supplement of KSM-66 is easily accessible, cost-
effective and has Informed Sport certification, making it safe to ingest without the risk of 
contaminated ingredients. The supplement is already highly used in the area of sports 
performance; importantly, this study aims to verify whether short-term supplementation has 
benefits and will inform the wider population on safe dosage guidelines. This benefits not only 
athletes, but practitioners on best procedure for their patients or clients. Weekly gym sessions 
are an integral part of footballer’s training regime, and if benefits are found in muscle strength, 
RPE and wellness scores of sleep quality, muscle soreness, stress and fatigue, athletes may 
wish to explore supplementation with KSM-66 to improve their strength output, perception 
of difficulty, sleep quality and muscle soreness with a safe method. Additionally, if athletes or 
active individuals experience high levels of stress and it affects their performance and 
recovery as a result, the study is applicable as the intervention may potentially improve these 
areas. 
 
Limitations 
Regarding participants, accuracy of food reporting and following supplementation guidelines 
will be based on trust between the author and participant. The study has convenience 
sampling, which allows for ease of data collection but lacks generalisability in the sample. The 
focus of the study is on subjective results, without the validity of physiological tests that could 
measure metabolic markers, such as cortisol, from blood samples. There is also a lack of 

 
23 
previous research on ASH and exercise in females and athletes, which impacts result 
credibility. 
 
Dissemination plan 
Full transparency of the measurements will be published within each of the corresponding 
three papers. No measurements or scores will be omitted. The purpose of the outreach is to 
contribute to the scientific knowledge of ASH and its effects on muscle strength and recovery 
in female athletes. Additionally, this research will provide scientific knowledge to sports 
science and the industry of sports nutrition of the influence of short-term ASH, and whether 
it provides any benefits to female athletes. The methods of sharing the scientific knowledge 
will include presentations at conferences, exhibitions, social media and published articles in 
journals.  
 
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30 
Appendix 
Sample Size Calculation 
 

 
31 
 
 

 
32 
Borg Scale 
 
 
 
 
 
 
 
 
 
 
 
 
 
(Vera & Rodríguez, 2022) 
 
 

 
33 
Total Quality Recovery questionnaire 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
(Martín, 2017; Francisco et al., 2020) 
 
 

 
34 
Hooper Index questionnaire 
 
 
 
 
 

 
35 
Supplement satisfaction questionnaire 
 
 
 
 
 
 
 
 
 
(Kuehl et al., 2010b) 
 
 
ASH

 
36 
Agreement document from club 
 
 

 
37