Independent and Synergistic Effect of Spirulina Maxima With Exercise on General Fitness, Lipid Profile and Antioxidant Capacity in Overweight and Obese Subjects
Phase: NA
Conditions: Cardiovascular Diseases
Sponsor: Universidad Autonoma de Ciudad Juarez

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Hypolipidemic and Antioxidant Capacity of Spirulina and Exercise 
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NCT number: NCT02837666. 
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Date: April 27, 2017 
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Responsible: Dr. Arnulfo Ramos Jiménez 
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Autonomous University of Ciudad Juárez, México 
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e-mail: aramos@uacj.mx 
 
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Abstract 
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Introduction: In order to reduce cardiovascular diseases risk factors, a healthy 
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diet must include dietary antioxidants from different sources (e.g. Spirulina 
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maxima) and regular practice of exercise should be promoted.  
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There is a little evidence from animal studies that Spirulina maxima and exercise, 
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decreases cardiovascular diseases risks factors. However, very few studies have 
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proven the independent or synergistic effect of Spirulina maxima plus exercise in 
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humans. Therefore, this study pretends to address this independent and 
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synergistic effect in overweight and obese subjects participating in a systematic 
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physical exercise program at moderate intensity on general fitness, plasma lipid 
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profile and antioxidant capacity in overweight and obese subjects. 
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Methods and analysis: Through a randomized, double-blind, placebo-controlled, 
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and a counterbalanced crossover study design, eighty healthy overweight, and 
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obese subjects will be evaluated during a twelve-week isoenergetic diet, 
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accompanied by 4.5 g a day of Spirulina maxima intake and/or a physical 
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systematic exercise program at moderate intensity. Body composition, oxygen 
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uptake, heart rate, capillary blood lactate, plasma concentrations of triacylglycerols, 
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total, low and high-density lipoprotein cholesterol, antioxidant status, lipid oxidation, 
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protein carbonyls, superoxide dismutase, catalase, glutathione, glutathione 
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peroxidase, glutathione reductase, and paraoxonase will be assessed.  
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Ethics and dissemination: This study and all the procedures have been approved 
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by the Universidad Autonoma de Ciudad Juarez Bioethics Committee. Moreover, 
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findings will be disseminated through peer-reviewed journals, national and 
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international conferences. 
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Trial registration number: ClinicalTrials.gov: NCT02837666. 
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Keywords: Spirulina, dyslipidemias, oxidative stress, exercise, body fat, 
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antioxidant. 
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Strengths and limitations of this study 
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• Working with obese people, one of the main risk factors for cardiovascular 
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diseases. 
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• This study can be extrapolated to different populations. 
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• Double-blind randomized controlled trial. 
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• There are not possible limitations to perform all the procedures described in 
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the present paper. 
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Background 
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Cardiovascular diseases (CVD) are the leading causes of death worldwide. Each 
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year, 17.5 million people die because of these diseases 1. Dyslipidemias are a 
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predisposing factor for CVD and are characterized by high concentrations of 
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triacylglycerols (TAG), total cholesterol (TC), low-density lipoproteins cholesterol 
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(LDL-c), and low concentrations of high-density lipoproteins cholesterol (HDL-c) 2. 
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Besides dyslipidemias, oxidative stress (OxS) is also observed to rise in obesity 3, 
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4, and it is also a predisposing factor for CVD 5- 8.  
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As a way to reduce OxS, dyslipidemias and the CVD incidence, it has been 
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proposed the intake of antioxidants that come from fruit and vegetable-rich diet or 
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nutritional supplements, mainly by unprocessed foods 9, 10. In this sense, 
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cyanobacterium Spirulina is an important source of antioxidants, currently 
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associated with cardiovascular protection properties 9, 11. 
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For centuries, Spirulina maxima (S. maxima) has been cultivated and used as a 
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nutritional supplement due to its content of amino acids and essential fatty acids, 
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vitamin C, vitamin E, tocopherols, and phycocyanins 12. Recently, S. maxima 
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studies have focused in verifying the biological activity of its components, including 
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hypolipidemic and antioxidant effects 13-15. However, most studies have been 
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conducted in animal models, with only a few studies focused on the biological 
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effect in humans. 
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Furthermore, it is known that the practice of systematic physical exercise 
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ameliorates CVD risks 10, 16; and it has been observed that also plays a role in OxS, 
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especially physical exercise at high intensity (PEHI) 17. PEHI considerably 
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increases general metabolism, oxygen uptake (VO2), and mitochondrial activity; 
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thus increasing the reactive oxygen species (ROS) production18. Instead, physical 
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exercise of moderated intensity (PEMI) has the best protective effect exerted 
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against CVD, mainly due to physiological adaptations, including the expression of 
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antioxidant enzymes 19, which stop the formation and propagation of ROS, and 
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improving redox status of the organism 20, 21. 
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Algae and cyanobacteria have shown promising pharmacological properties 
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providing health benefits and physical improvements 22. These effects are 
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attributed to their specific profile in bioactive compounds such as uncommon 
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carotenoids, phenolic compounds, tocopherols besides their well-known content in 
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vitamins and good quality proteins associated to their specific chemical structure 
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and interaction with biological membranes 23. 
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In this sense, there is evidence that the cyanobacteria Spirulina, in addition to 
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exercise, lower CVD risks. This was mainly observed in animal models 24-26. 
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However, no studies in humans under Spirulina and exercise experimental designs 
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proving these benefits have been reported yet. Therefore, the ISESE study will 
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analyses the independent and synergistic effect of the intake of S. maxima with a 
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PEMI on general fitness, plasma lipid profile, and redox status in overweight and 
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obese subjects.  
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Methods/Design 
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Hypothesis 
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Spirulina maxima intake and a dosed physical activity program will improve lipids 
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profile, general fitness and antioxidant status in overweight and obese subjects. 
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Objectives 
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The ISESE project’s main objective is to demonstrate that ingestion of S. maxima 
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and a dosed physical activity program will decrease, both independently and 
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synergistically, some CVD risks in overweight and obese subjects.  
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Outcome measures 
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The primary outcome measure will be changes in the plasma lipid profile after a 
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six-week of treatment (plasma TAG, TC, HDL-c, and LDL-c by using standardized 
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enzymatic methods). 
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The secondary outcome measure will be changes in general fitness (maximal 
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oxygen uptake (VO2 max), heart rate (HR), in onset blood lactate accumulation, 
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and body fat mass); as well as changes in redox status (malondialdehyde (MDA), 
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protein carbonyls, paraoxonase (PON1), superoxide dismutase (SOD), catalase 
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glutathione (GSH), glutathione reductase (GR), and glutathione peroxidase (GPx). 
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Participants’ eligibility 
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The inclusion criteria will be the age between 18 and 45 years old of both sexes: 
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40 overweight (Body mass index (BMI): 25-29.9 kg/m2) and 40 obese (BMI: >30 
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kg/m2). The subjects´ exclusion criteria will be: drinking more than 100 mL of 
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alcohol a week, taking drugs and/or food or vitamin supplements, having a chronic 
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disease, or having a physical or electrocardiographic injury that prevents them from 
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engaging in any regular physical exercise. The elimination criteria will be that the 
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program subjects´ attendance will be lower than 80% and/or 20% change in their 
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body weight during study. 
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Call to participation, ethics and safety procedures 
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University students of Ciudad Juarez, Mexico with overweight and obesity will be 
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personally invited to participate. Participants will be informed of the purpose of the 
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study, and of the physical, clinical and biochemical procedures, benefits, physical 
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intensity tests, and risks, all before beginning the clinical trial. To guarantee the 
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good physical and mental health of the participants at the beginning of all the 
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studies, a medical clinical laboratory study examination and an electrocardiography 
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at rest tests will be performed. Noting that investigators are trained in 
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cardiopulmonary reanimation and that the laboratory has the necessary 
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communication tools and channels to perform emergency procedures. This study 
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and all the procedures have been registered appropriately in ClinicalTrials.gov 
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database (NCT02837666) and approved by the Bioethics Committee of the 
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Universidad Autonoma de Ciudad Juarez (Supplementary files). That participants’ 
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acceptance formalized by means of written informed consent and their anonymity 
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and confidentiality will be strictly enforced. This study will be conducted in 
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compliance with the principles of the Declaration of Helsinki. 
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Baseline measurements 
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A day before beginning the treatment, subjects will visit the laboratory for baseline 
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measurements. Body mass will be measured with subjects lightly dressed and 
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barefoot in an electronic balance, and standing height with a stadiometer. Body fat 
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percentage will be measured by plethysmography method (BodPod, USA), electric 
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bioimpedance (in body 160, US), and by dual-energy X-ray absorptiometry (DXA) 
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(Prodigy v6.8; GE Lunar, Milwaukee, WI) according to manual guidelines. 
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Anthropometric parameters will be taken by the standardized method of the 
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International Society for the Advancement of Kinanthropometry (ISAK), and 
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according with ISAK Anthropometry Accreditation protocols 27. Researchers are 
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standardized in handling equipment and procedures. 
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Study design 
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The study consists of S. maxima treatment during 14 weeks in a randomized, 
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double-blind, placebo-controlled, and counterbalanced crossover design, aimed to 
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eliminate inter-individual bias as well as boosting the reliability of final results; 
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moreover, this design is commonly used in clinical trials in order to have better 
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results and conclusions 28. Eligible participants (n= 80) will be divided into two 
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groups, one with systematic practice of physical exercise (GEx) and other one 
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without it (GNEx). The participants of each group will be randomized in equal 
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proportions between S. maxima and placebo treatment, receiving either 4.5 g daily, 
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in a non-transparent capsule during 12 weeks (6 weeks for the first treatment - 2 
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weeks of washout period - 6 weeks for the second treatment) (Figure 1). Before 
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any reported allergic reaction occurs, the treatment will be discontinued. 
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To maintain the overall quality and legitimacy of the clinical trial, code interruptions 
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should occur only in exceptional circumstances when knowledge of the actual 
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treatment is essential for further management of the participant. Investigators are 
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encouraged to discuss with a Medical Advisor if they believe that unblinding is 
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necessary. Unblinding should not necessarily be a reason for study 
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discontinuation. 
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A student outside the research team will feed data into the computer in separate 
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datasheets, so that the researchers can analyze the data without having access to 
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information about the allocation treatments, storing all the participants’ files in 
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numerical order in a safe and accessible place. The participants´ files will be stored 
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for a period of five years after the end of the study. 
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The sample size was determined by using the statistical program G*Power 29, 
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selecting a sample of 73 subjects with α = 0.05 and p = 0.85. This sample will be 
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fixed to 80 subjects in order to anticipate the possible desertions from the study. 
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Maximum intensity tests 
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Each subject will participate in four stress tests performed at maximum intensity 
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(MIT) during the study; during MIT record, consumed O2 and produced CO2 will be 
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taken by a gas analyzer (Cortex MetaLyzer® 3B, Germany), HR with a Polar H7 
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sensor (Polar Electro, Lake Success, NY), and lactate in capillary blood samples 
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will be assayed in a YSI 1500 Sport Lactate Analyzer (YSI Life sciences, OH, 
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USA). The MIT protocol consists of using a cycle ergometer (Monark ergomedic 
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828 E; Monark exercise AB, 105 Vansbro, Sweden) initiating with a workload of 50-
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75 W with increments of 15-30 W every 3 min for until 15 min or when the subject 
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could no longer pedal more than 40 revolutions/min, finishing the test when 
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reaching 90-100 % of the maximum HR reserve; MIT will not be valid for less than 
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9 min (Figure 2). At the end of each increment load (3 min), capillary blood 
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samples will be taken to determine lactate, and the physical perceived effort will be 
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registered by using the Borg scale. Measurements of HR, glucose and blood 
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pressure will serve to take care of the subject’s health during each MIT. 
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Before the first MIT, subjects will decide to be in the GEx or GNEx, and then they 
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will be divided randomly for the treatment, which consists in a daily dosage of 4.5 g 
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of Spirulina or placebo in capsules which will be recommended the subjects to 
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consume them before each meal every day during six weeks. The day after the 
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first supplementation period, subjects will come back to perform the second MIT 
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with identical conditions to the first one. The third MIT will be performed after a 
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washout period of two weeks to remove the effects of treatment and avoid any 
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possible delayed effect of S. maxima in the organism. The last MIT will be 
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performed after the second treatment with identical conditions to the previous three 
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times. General fitness parameters will be measured the same day of all the MITs to 
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have accurate results.  
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Randomization 
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All subjects who consent to participation and who meet the inclusion criteria will be 
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randomly assigned by an investigator who will not have any other interference in 
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the clinical study; he will create a database with all the treatments through the 
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duration of the study. Therefore, randomization will be carried out without any 
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influence of the principal investigators. Participants will be randomly assigned to S 
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maxima or placebo treatment with a 1:1 allocation according to a computer-
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generated random schedule stratified by site and the baseline score of the Action 
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Arm Research Test using permuted blocks of random sizes with an online, central 
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randomization service (TENALEA). Block sizes will not be disclosed, to ensure 
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concealment. 
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Adherence assessments 
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Multiple methods will be used to assess treatment adherence including pill 
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counting; reasons for non-compliance, and use of the capsules. Participants will 
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return each week to receive new capsules. All treatment data will be recorded on 
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the appropriate case report form. 
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Dietary analysis 
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All participants will be subjected to a nutritional survey to define the daily calories 
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required to establish a custom diet. Dietary intake will be monitored through 
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retrospective methods including the 24 h recall (which investigates intake over a 
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specific day), and the food frequency questionnaire (a summary of usual intake of 
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different categories of foods). These accurate and validated techniques allow 
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quantifying the types and quantities of foods and beverages consumed during the 
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period of interest in the past. Dietary intake evaluations will be performed at each 
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MIT of the study in order to record the possible variability in food consumption 
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patterns. 
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Physical exercise protocol 
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General health assessment and physical activity questionnaires (PAR-Q and YOU) 
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will be conducted; this will be done to ensure that there are no physical 
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impediments to exercise. GEx participants are going to exercise five days a week 
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with the following protocol: between 5 and 10 min of warm-up exercise, between 
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20-30 min muscular endurance exercise and 20-30 minutes of aerobic exercise 
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(cardiovascular exercise): walking, jogging, running and/or cycling. Three days a 
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week aerobic intensities will be between 60% and 80% and two days between 70% 
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and 90% of the maximum HR reserve, and five final minutes of stretching.  
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Sample collection and biochemical analysis 
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Blood samples (8 mL) will be collected before and after each MIT from the 
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antecubital vein into ethylenediaminetetraacetic acid (EDTA) tubes after 10-12 h of 
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fasting, plasma from blood samples will be obtained by refrigerated centrifugation 
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(4°C) at 5 000 g for 10 minutes. Plasma glucose TC, HDL-c, LDL-c, and TAG 
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concentrations will be analyzed by using standard enzymatic procedures (Jas 
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Diagnostics, Inc. Mexico) with a spectrophotometer (Genesys 10 UV; Thermo 
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Electron Corporation, USA). Plasma will be used to measure MDA and GSH 
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content and activity of GPx and GR according to Jimenez-Osorio et al. 30, total 
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antioxidant status (TAS) by 2,2-diphenyl-1-picrylhydrazyl (DPPH) technique as 
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previously described by Koren et al. 31, oxygen radical absorbance capacity 
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(ORAC) following Prior et al. 32 methodology. The erythrocytes will be washed with 
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cold 0.9% saline solution twice and are going to be used for SOD and catalase 
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activity assays as previously described by Jimenez-Osorio et al. 30, and plasma 
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PON1 content will be measured by following Perez-Herrera et al. 33 methods. 
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Statistical analysis 
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To evaluate data distribution, normality and homoscedasticity tests on each of 
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response variables analyzed will be conducted. In order to analyze differences 
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between variables before and after the study and between the groups, an ANOVA 
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test will be performed. To analyze possible associations between variables, a 
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bivariate correlation analysis will be used. To analyze independence between 
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variables, a multiple regression analysis will be performed. The software to use is 
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GraphPad Prism 6. 
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Discussion 
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Cardiovascular diseases are the leading cause of mortality worldwide, with OxS, 
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overweight, obesity, and dyslipidemias being the main risk factors. Many drugs 
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aimed at reducing these risk factors have often collateral side effects when used, 
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and many people often use complementary medicine instead. For this and other 
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reasons, the pharmacological industries have focused on developing new 
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treatment options34; S. maxima research has proven benefits to reduce CVD risk 
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factors, isolated or in combination with systematic practice of physical exercise, 
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mainly in animal models 9, 35, 36. 
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This study is summarized and described in experimental design, procedures, 
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compliance with ethical principles, and statistical analyses. Since the double-blind 
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design, compressively described the methodology and a higher number of 
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participants than other studies, it is expected to get a better support for the 
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hypothesis that S. maxima will have hypolipidemic and antioxidant effect against 
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intense exercise and obesity. It is noteworthy that, not every overweight or obese 
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patient has issues with lipid metabolism, nowadays the association between the 
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augmentation of body fat and lipid disturbances is well known 37; for that reason, 
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the primary focus of this protocol is prevention and it justifies why we do not 
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consider dyslipidemia as an inclusion criterion; even though, they could present 
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this condition. 
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Finally, it is known that systematic practice of exercise and a balanced diet not only 
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favors reduction of weight in obese people, but it generally decreases 
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cardiometabolic risks 2, and thus it probably generates an increase in antioxidant 
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capacity 38; however, no studies on the metabolic oxidation decreased by 
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administration of Spirulina in people with obesity were identified. In conclusion, 
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well-designed trials are needed to clarify the value of S. maxima supplementation 
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in clinical practice 39and its complementary effect with or without exercise against 
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dyslipidemias and OxS in overweight and obesity, a fact practically unknown at this 
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time40. The later establishes the importance of this study. 
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Competing interests 
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The authors declare that they have no competing interests. 
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Funding 
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This research will be funded by Programa para el Desarrollo Profesional Docente, 
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SEP, México; and by Universidad Autonoma de Ciudad Juarez, Chihuahua, 
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Mexico. Study design; collection, management, analysis, interpretation of data; and 
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the decision to submit the results for publication will be/are all independent of the 
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funders. Ultimate authority for these activities will lie with principal investigators. 
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Author affiliations 
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1Departamento de Ciencias Químico Biológicas, Instituto de Ciencias Biomédicas. 
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Universidad Autónoma de Ciudad Juárez, Ciudad Juárez, Chihuahua, México. 
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2Departamento de Ciencias de la Salud, Instituto de Ciencias Biomédicas. 
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Universidad Autónoma de Ciudad Juárez, Ciudad Juárez, Chihuahua, México. 
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3Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional 
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Autónoma de México, Ciudad de México.  4Facultad de Ciencias de la Cultura 
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Física, Universidad Autónoma de Chihuahua, Chihuahua, México. 5 Departamento 
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de Biología, Facultad de Química, Universidad Nacional Autónoma de México, 
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Ciudad de México. 
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Contributors 
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All authors contributed to the development of the study protocol and to the critical 
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revision of the paper and approved the final version. RPHT, ARJ, and JALD will be 
295 
involved in patients’ recruitment. MAHL and AWM will analyze the data, MAHL and 
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MAJO will do the lipid profile analyses, RUR, ARJ and MAHL will do the body 
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composition measurements of all patients, LARC and JPC will do all the redox 
298 
status analysis. 
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Acknowledgements 
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We want to thank the Universidad Autonoma de Ciudad Juarez Institutional 
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Bioethics Committee for the interest in this study, approving it and all its 
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procedures. Also, we thank the Consejo Nacional de Ciencia y Tecnologia 
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(CONACyT) by providing a scholarship to the first author. 
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Figure 1. Experimental design for independent and synergistic effect of Spirulina 
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maxima and exercise. Same color means the same group of participants. 
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Figure 2. Design of maximum intensity tests. ↓ indicates the interval at which the 
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resistance will be increased; in addition, physical perceived effort, heart 
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rate, and capillary blood samples will be measured. 
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