Introduction: In sport science it is well known that individual diet has an important role in affecting a person’s performance [1]. In this regard, in the last 10-20 years energy and sport drinks have become increasingly popular among elite and amateur athletes. The worldwide consumption of energy drinks increased in 2006 to 906 million gallons [2]. Just in Brazil, from 2008 to 2010, the production of energy drinks doubled [3]. A possible reason for their popularity is that they claim to help enhance physiological performance by replenishing the electrolytes lost during exercise and by boosting the body’s energy demands [4]. Although this claim is still under debate the effect of energy drinks may be quite different depending on the type of activity undertaken by athletes and it may be highly individual [5]. It is important to determine which changes in the sweat profiling are triggered by the consumption of different types of energy drinks and link those to sport performance. Our attention focuses on the role of caffeine and sodium since these two are common components in sport drinks. Although caffeine is widely part of daily diet, it is considered to be a drug and its level in urine has to be less than 15 µg mL-1 during sport competitions [6,7]. Electrolyte balance and adequate hydration levels have an important role in avoiding heat-related illness and can have an overall effect on enhancing or impairing human sports performances [8,9]. However, it is difficult to advise an optimum sodium intake suitable for all since sweat sodium concentrations and sweat rates vary greatly between individuals [10,11]. Aim: Sweat contains a range of clinically relevant metabolites and is easily accessible for analysis. This work investigated the effect of different sports drinks on sweat composition in order to explore the physiological effects of these drinks. This type of technology is essential to meet the demands for non-invasive analysis of biological media in the biomedical field to monitor health and in sport science to assess physiology and performance [12]. Methodology: Two different sports drinks were chosen for this study, Hi Five Extreme Energy Source, and Precision Hydration sports drink. Bottled water was used as a control. The two sports drinks were chosen as they have large differences in caffeine and sodium content. Objective: To evaluate the effect of these three drinks on sweat composition. High-performance liquid chromatography (HPLC) methods were employed to determine the concentration of the caffeine in sweat samples. Flame-photometry and in-house built sodium sensors have been employed to detect sodium levels. Results and Discussion: At the first stage some energy drinks were chosen, the caffeine and sodium content were compared with those on the label and it was confirmed that the values are similar. After, preliminary test with a subject, the HPLC chromatogram for caffeine showed overlapping peaks which did not permit the identification of caffeine peak. In view of all the issues, in order to establish a new method in the HPLC system three different mobile phases were tested and the best one was 30% methanol and 70% water where the peak can be fully resolved between 1,75 and 1,85 minutes.