Effects of ingesting a pre-workout dietary supplement with and without synephrine for 8 weeks on training adaptations in resistance-trained males

Apparently healthy, resistance-trained males were recruited to participate from local advertisements. Inclusion criteria required that each participant have at least 6 months of resistance training immediately prior to entering the study inclusive of performing bench press and leg press or squat. Participants were excluded if they presented with a history of treatment for metabolic disease, hypertension, thyroid disease, arrhythmias, and/or cardiovascular disease; and/or were currently using any prescription medication. Further exclusion criteria included an intolerance to caffeine and/or other natural stimulants; a history of smoking; and, excessive alcohol consumption (>12 drinks/week).

A total of 213 individuals responded to advertisements to participate in this study. Participants who met initial study entry criteria via phone interview or online questionnaire screening were invited to a familiarization session where the details of the study were explained, informed consent was obtained, medical history was assessed, a fasting blood sample was obtained, and a general medical exam was performed by a registered nurse to determine eligibility to participate in the study. A total of 122 individuals were cleared to participate in the study and participated in a familiarization session. This included explanation of the protocol, instructions for completing the food record forms and training program logs, and practicing the strength and anaerobic capacity tests that were used in the study. Participants were then matched for age, body mass, and fat free mass (FFM) and randomized into one of three dietary supplement intervention arms in a randomized manner. A total of 80 males (22 ± 4 y, 178 ± 6 cm, 80.9 ± 13.9 kg, 15.2 ± 0.7% fat, 25.6 ± 4.0 kg/m²) completed the study.

All participants were required to follow the same resistance training routine. The resistance training program consisted of training 4-days per week split into two upper and two lower body workouts per week primarily consisting of free-weight exercises for a total of 8-weeks. The 8-week training protocol was periodized in 2–3 week segments and consisted of selection from a list of 2–4 exercises for the following muscle groups: chest (two exercises for a total of six sets), back (two exercise for a total of six sets), shoulders (one exercise for a total of three sets), biceps (one exercise for a total of three sets), triceps (one exercise for a total of three sets), abdominals (one exercise for a total of three sets), quadriceps (two exercises for a total of six sets), hamstrings (two exercises for a total of six sets), and calves (one exercise for three sets). Each exercise consisted of three sets of ten repetitions (week 1–3), eight repetitions (week 4–6), or six repetitions (week 7–8) performed with as much weight as the participant could perform per set. The participants recorded the amount of weight lifted during each set of exercise on training log. A training partner or fitness instructor provided signed verification that the work out was completed as recorded. Prior research from our lab has shown that this program is effective in promoting significant strength and fat free mass gains in resistance-trained athletes without nutritional intervention [45].

Participants were matched for age, body mass, and FFM and randomly assigned to ingest in a double-blind manner either: (1) a flavored dextrose placebo (PLA); (2) a PWS containing beta-alanine (3 g), creatine nitrate as a salt (2 g), arginine alpha-ketoglutarate (2 g), N-Acetyl-L-Tyrosine (300 mg), caffeine (284 mg), Mucuna pruiriens extract standardized for 15% L-Dopa (15 mg), Vitamin C as Ascorbic Acid (500 mg), niacin (60 mg), folate as folic acid (50 mg), and 70 mg of Vitamin B12 as Methylcobalamin (Cellucor C4 Pre-Workout, Nutrabolt, Bryan, TX); or, 3.) the PWS with Citrus aurantium (PWS + S) extract standardized for 30% synephrine (20 mg) (Nutratech Inc., Caldwell, NJ). Supplements were independently packaged by a third party into coded single foil packets for double-blind administration following Good Manufacturing Practices and certified to contain the aforementioned ingredients by VMI Nutrition (Salt Lake City, UT). All supplements had similar color and powdered texture. Participants were instructed to ingest one foil packet per day approximately 15–30 min prior to exercise on training days and in the morning with breakfast on non-training days. Supplement compliance was verified by weekly compliance verification and collecting and counting empty packets.

Figure 1 shows the timeline of tests performed. Participants were instructed to refrain from exercise, caffeine, and supplements containing stimulants for 48-h prior to testing. Participants presented to the lab after a 12-h fast and were required to provide a 4-days food-log that recorded their consumption of food and energy containing fluids. Participants training logs were assessed by a trained exercise physiologist to ensure compliance and they submitted weekly side-effect questionnaires. Participants then donated ~ 20 ml of blood via venipuncture. Following blood sampling, we administered a series of tests. This included determination of body weight, total body water using bioelectrical impedance (BIA), body composition using dual-energy x-ray absorptiometry (DXA), resting heart rate and blood pressure, cognitive function (Stroop Word - Color test), perceptions of readiness to perform via use of a visual analogue scale (VAS), one repetition maximum (1 RM) bench press, 1 RM leg press, and a 30s Wingate anaerobic capacity test on a cycle ergometer. Subjects rested 5-min between bench press, leg press, and Wingate tests as well as 2-min between sets on the bench press and leg press. Participants completed these assessments at 0, 4, and 8-weeks of training.

Body mass and height were determined according to standard procedures using a Healthometer Professional 500KL (Pelstar LLC, Alsip, IL, USA) self-calibrating digital scale with an accuracy of ± 0.02 kg. Total body water (TBW) was measured using bioelectrical impedance analysis (ImpediMed DF50, San Diego, CA) using standard procedures. Whole body bone density and body composition measures (excluding cranium) were determined with a Hologic Discovery W Dual-Energy X-ray Absorptiometer (DEXA; Hologic Inc., Waltham, MA, USA) equipped with APEX Software (APEX Corporation Software, Pittsburg, PA, USA) by using procedures previously described [8, 50]. Mean test-retest reliability studies performed on male athletes in our lab over repeated days revealed mean coefficients of variation (C_v) for total bone mineral content and total fat free/soft tissue mass of 0.31–0.45% with a mean intraclass correlation of 0.985 [51]. On the day of each test, the equipment was calibrated following the manufacturer’s guidelines.

As soon as the DXA scan was completed (about 6-min), resting heart rate was determined in the supine position by palpitation of the radial artery using standard procedures [52]. Blood pressure was then assessed by auscultation of the brachial artery using a mercurial sphygmomanometer using standard clinical procedures [52].

Cognitive function was assessed using the Stroop Word-Color test standardized by Golden [53]. The test consists of three pages/tests with 100 items, presented in 5 columns of 20 items. Items on the first page (Word) are the color words RED, GREEN, and BLUE in black ink. On the second page (Color) the items are XXX’s colored in red, green, or blue ink. Items on the third page (Word-Color) are the words RED, GREEN, and BLUE printed in red, green, or blue ink with the limitation that word and ink could not match. Participants were given standardized instructions and asked to read aloud each word or color on each page as fast as they could for 45 s. The number of correct responses obtained on each test during the time period is used to assess cognitive function.

Perceptions about readiness to perform were assessed using a visual analogue scale (VAS) using a 5-item descriptive scale (strongly disagree, disagree, neutral, agree, strongly agree) arranged on a 20 cm dotted bar with these terms equidistant along the scale. Participants were asked to respond to the following questions; “I slept well last night”; “I am looking forward to today’s workout”; “I am optimistic about my future performance”; “I feel vigorous and energetic”; “My appetite is great”; and, “I have little muscle soreness”. Participants circled the number or dot between numbers that best described their current perceptions related to these questions.

Strength tests were performed using an isotonic Olympic bench press (Nebula Fitness, Versailles, OH) according to standard procedures [45]. Participants followed a warm-up consisting of 10 repetitions using 50% of their estimated 1RM, 5 repetitions using 70% of their estimated 1RM, and 1 repetition using 90% of their estimated 1RM. Participants were given 2-min recovery between attempts and performed 1RM lifts until reaching a failure weight. After 5-min recovery, participants warmed-up in a similar fashion as described above and then performed 1RM lift attempts on a standard hip sled/leg press (Nebula Fitness, Versailles, OH) according to standard procedures [45]. Test to test reliability of performing these tests in our lab on resistance-trained participants have yielded low C_v’s and high reliability for the bench press (1.9%, r = 0.94) and hip sled/leg press (0.7%, r = 0.91).

Prior to performing the anaerobic capacity test, participants warmed-up on a bicycle ergometer at a self-selected work rate. Wingate anaerobic capacity tests were performed using a Lode Excalibur Sport Ergometer (Lode BV, Groningen, The Netherlands) with work rate set at of 7.5 J/kg/rev. Participants were asked to pedal as fast as possible prior to application of the workload and sprint at an all-out maximal capacity for 30s. This test measures absolute and relative peak and mean power and total work. Test-to-test variability in performing repeated Wingate anaerobic capacity tests in our laboratory yielded a C_v of 15% with a test retest correlation of r = 0.98 for mean power [47]. Participants practiced the anaerobic capacity test during the familiarization session to minimize learning effects.

All blood samples were analyzed for standard blood chemistries inclusive of alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), creatinine, blood urea nitrogen (BUN), creatine kinase (CK), lactate dehydrogenase (LDH), glucose, and blood lipids (total cholesterol, high density lipoprotein [HDL], low density lipoprotein [LDL], triglycerides [TG]) using a Cobas® c 111 (Roche Diagnostics, Basel, Switzerland). The internal quality control for the Cobas® c 111 was performed according to standard procedures [54] using two levels of control fluids purchased from the manufacturer to calibrate to acceptable SD’s and C_v’s. Samples were re-run if the observed values were outside control values and/or clinical norms according to standard procedures. Test-to-test reliability assessment of assays evaluated in this study yielded mean C_V’s < ±2.0% with r values > 0.99. We also assessed a complete blood count with platelet differential on whole blood (hemoglobin, hematocrit, red blood cell counts, mean corpuscle volume (MCV), mean corpuscle hemoglobin (MCH), mean corpuscle hemoglobin concentration (MCHC), red cell distribution width (RDW), white blood cell counts, lymphocytes, granulocytes, and mid-range absolute count (MID) using a Abbott Cell Dyn 1800 (Abbott Laboratories, Abbott Park, IL, USA) automated hematology analyzer. The internal quality control for Abbott Cell Dyn 1800 was performed using three levels of control fluids to calibrate to acceptable SD’s and C_v’s. Test-to-test reliability assessment of assays evaluated in this study yielded mean C_V’s < ±6.3% with r values > 0.9.

Baseline demographic and training volume data were analyzed by one-way analysis of variance (ANOVA). All data were analyzed using general linear models (GLM) multivariate analysis of variance (MANOVA) with repeated measures with Wilks’ Lambda and Greenhouse-Geisser adjustments. For performance and cognitive function data, baseline values were used as a covariate and run with MANOVA for repeated measures with differences between groups assessed using a Dunnet-Hsu post-hoc assessment vs. the PLA condition. These data were also graphed with means and 95% Confidence Interval (CI) to determine whether changes from baseline were significant [55]. We also analyzed the number of changes in blood chemistry values observed from normal to exceeding normal clinical limits from baseline to week 4, baseline to week 8 and week 4 to week 8 using a Chi-square analysis to examine whether any nutritional treatment promoted a significant increase in the number of participants with values exceeding normal. All data are presented as mean ± SD or mean change and 95% CI.

Table 1 presents participant demographics by group assignment. A total of 80 participants completed the study (PLA = 27, PWS = 27, PWS + S = 26). One-way ANOVA revealed that no significant differences among groups in baseline age, height, body weight, or body mass index.

Table 2 presents total training volume observed among groups for upper and lower extremity exercises. One-way ANOVA analysis revealed that there were no significant differences in lifting volumes among groups. Table 3 shows 4-day diet analysis data observed among groups at 0, 4, and 8 weeks of training. MANOVA analysis revealed that no significant group x time interactions were observed among groups in relative energy intake (p = 0.19), protein intake (p = 0.72), carbohydrate intake (p = 0.55) or fat intake (p = 0.79).

Reported frequency and severity of dizziness, headaches, racing heart rate, palpitations, shortness of breath, nervousness, blurred vision, and/or other symptom were so infrequent among participants that statistical analysis was not valid as the vast majority of participants (i.e., 90–98%) typically reported 0 ratings on each item throughout the study. No study participant required medical referral.

Table 4 presents body composition data observed during the course of the study. MANOVA analysis revealed significant time effects in changes in body weight (0.96 ± 2.6 kg, p = 0.003) and FFM 0.67 ± 1.8 kg, p = 0.001). However, no significant interactions were observed among groups in body weight (p = 0.28), fat mass (p = 0.61), FFM (p = 0.28), body fat percentage (p = 0.36), or percent total body water (p = 0.37).

MANOVA analysis revealed no significant differences among groups in hemodynamic responses during the study (Wilks’ Lambda group p = 0.62, time p = 0.33, and group x time p = 0.87). Univariate analysis revealed no indication that PWS or PWS + S supplementation increased resting heart rate (p = 0.26), systolic blood pressure (p = 0.96), or diastolic blood pressure (p = 0.54) during training compared to the PLA group. All group means remained within ± 2 beats/min for heart rate and ± 2 mmHg for blood pressure from baseline values.

Table 5 shows the results for cognitive function testing. MANOVA analysis revealed Wilks’ Lambda overall time effects (p < 0.001) with no significant interaction effects (p = 0.17). MANOVA univariate analysis showed similar trends. However, univariate ANOVA analysis revealed an interaction trend (p = 0.087) among groups in color responses and a significant quadratic effect among groups in Word-Color counts (p = 0.04). Post-hoc analysis revealed that the PWS group demonstrated the greatest change in color counts from baseline while changes in word-color counts were seen sooner in the PWS and PWS + S groups (4-weeks) compared to the PLA group. Further, MANOVA analysis using baseline values as a covariate revealed some differences among groups in changes in cognitive function. As can be seen in Fig. 2, mean changes in Color, Word, and Word-Color counts where generally increased to a greater degree with 95% CI’s above baseline in the PWS and/or PWS + S groups compared to the PLA group values that were lower and had 95% CI’s crossing baseline. After 8-weeeks of intervention, all groups demonstrated significant increases in Color, Word, and Color-Word counts. More specifically, comparisons at week 4 demonstrated a significant increase in Word count for the PLA (3.92 counts, 95% CI .39, 7.45) and PWS + S (5.46 counts, 95% CI 2.09, 9.19) group, but not for PWS (3.21 counts, 95% CI −0.31, 6.72). By week 8, all groups increased their respective word counts: PLA (6.74 counts, 95% CI 3.32, 10.16), PWS (7.56 counts, 95% CI 4.15, 10.97) and PWS + S (9.93 counts, 95% CI 6.49, 13.73). For the Color assessment comparison, week 4 changes are: PLA (2.77 counts, 95% CI, 0.43, 5.09); PWS (5.05 counts, 95% CI, 2.72, 7.38); and, PWS + S (2.57 counts, 95% CI, 0.24, 4.88). For week 8, color assessment changes are: PLA (4.90 counts, 95% CI, 2.32, 7.46); PWS (8.33 counts, 95% CI, 5.76, 10.89); and, PWS + S (5.08 counts, 95% CI, 2.51, 7.63). Week 4 word-color changes were significant for the PWS (3.99 counts, 95% CI, 1.75, 6.23), and PWS + S (5.27 counts, 95% CI, 3.01, 7.52), but not the PLA (2.08 counts, 95% CI, −0.15, 4.31) group. By week 8, all groups demonstrated a significant increase in word-color counts: PLA (5.02 counts, 95% CI, 2.44, 7.59); PWS (5.84 counts, 95% CI, 3.27, 8.41); and, PWS + S (6.13 counts, 95% CI, 3.54, 8.72).

Table 6 presents Readiness to Perform VAS data observed throughout the study. MANOVA revealed an overall time effect with no significant interactions among groups. Likewise, MANOVA using baseline values and age as a covariate and analysis of mean changes with 95% CI’s revealed no significant differences among groups.

Performance testing outcomes are presented in Table 7. MANOVA revealed significant time effects for bench press and leg press 1RM performance. However, no significant univariate interactions were observed among groups. MANOVA analysis using baseline values as a covariate and assessment of mean change and 95% CI’s of 1RM strength data (Fig. 3) revealed that there were significant increases in bench press 1RM strength at week 4 for the PWS (8.17 kg; 95% CI 1.88, 14.47) and PWS + S (6.95 kg; 95% CI 0.62, 13.28), but not for the PLA (5.45 kg, 95% CI −0.82, 11.73). By week 8, all groups demonstrated a significant increase in BP 1 RM: PLA (7.18 kg, 95% CI 1.01, 13.36), PWS (14.36, 95% CI 8.13, 20.59) and PWS + S (13.84 kg, 95% CI 7.64, 20.04). No between group differences were noted at week 4 or week 8. A similar pattern for leg press 1 RM strength improvement was observed within the PWS (61.84 kg, 95% CI 24.96, 98.725) and PWS + S 44.89 kg, 95% CI 8.31, 81.47) groups, but not for PLA (36.50, 95% CI, −0-21, 73.2). Similarly, by week 8, all groups increased their LP 1RM as follows: PLA (43.28 kg, 95% CI 4.16, 82.41), PWS (79.23 kg, 95% CI 39.12, 118.54), and PWS + S (89.54 kg, 95% CI 50.55, 128.53). No between groups differences were noted at week 4 or week 8. Similar results were observed in Wingate anaerobic capacity assessment. MANOVA analysis using baseline values and ages as covariates revealed that a significant increase in Wingate peak power for PWS + S and PLA at week 4; yet no significant differences were otherwise noted at week 8 or among other Wingate parameters examined.

Tables 8, 9 and 10 show blood chemistry data analyzed during the study. MANOVA revealed some time effects in several variables indicative of individuals engaged in heavy resistance exercise training with no significant group x time interactions in muscle and liver enzymes, markers of catabolism, or blood lipids. Univariate ANOVA analysis revealed a significant quadratic effect in blood glucose values. Post-hoc analysis revealed the PLA group had a small but significant increase in blood glucose after 4-weeks of training while all groups were higher after 8-weeks of training. However, no significant differences were seen among groups and values remained within normal ranges. Table 11 shows Chi squared categorical analysis. No significant differences were observed among groups in the number of participants who observed changes in blood chemistry markers above normal baseline values.

The PWS’s investigated in this study contained 284 mg of caffeine, 300 mg of N-Acetyl-L-Tyrosine, and 15 mg of L-Dopa extracted from Mucuna pruiriens. Numerous studies have shown that ingesting caffeine (e.g., 3–6 mg/kg) can improve exercise performance, cognitive function, and/or vigilance [5]. In the present study, participants ingested an average of 3.5 mg/kg of caffeine prior to training sessions. There is also some evidence that tyrosine supplementation can improve cognitive function and/or memory [13, 14] although dosages typically studied (i.e., 50–150 mg/kg or 1.6–12 g) were much higher than the amount investigated in this study (i.e., 300 mg). Additionally, there is some evidence that L-Dopa supplementation (e.g., 150 mg) can affect cognitive function and/or reduce perceptions of fear or anxiety [15, 16] although the dosages studied in the present investigation are much lower. Thus, it is conceivable that ingesting a PWS with ergogenic levels of caffeine (and possibly tyrosine and/or L-Dopa) prior to exercise may provide some improvement in cognitive function and/or training adaptations due to a greater ability to focus on exercise training.

In the present study, analysis of mean changes from baseline with 95% CI’s (Fig. 2) indicated that change in Stroop Color and Word-Color counts were significantly above baseline in the PWS group and changes in Word count, Color count, and Word-Color counts were greater in the PWS + S group after 4-weeks of training and these changes were generally greater than the placebo group. These findings are consistent with several studies that reported that acute or chronic ingestion of PWS formulations containing caffeine positively affected cognitive function, concentration, and/or perceptions of energy [5, 56, 57]. The Stroop Color-Word test assesses attention, processing speed, and executive function which have been shown to decline as one fatigues. Theoretically, maintaining cognitive function during the latter stages of competition can improve performance particularly in events that require quick decision making. However, after 8-weeks of training and supplementation in the present study, all groups observed similar improvements in Stroop Word, Color, and Word-Color counts and no differences were observed in questions related to readiness to perform. These findings are consistent with other studies reporting no overall benefit of PWS supplementation on cognitive function, concentration, and/or perceptions of energy [58, 59] and suggest that the ergogenic value observed may lessen over time. However, it is also possible that since performing the 1 RM and anaerobic sprint capacity tests did not require significant executive or cognitive functioning to perform, there may be benefits on other performance tasks that require more decision making and attention.

The PWS’s examined in the present study contained a number of nutrients that have been purported to influence training adaptations. For example, the PWS’s contained 3 g/day of beta-alanine. Beta-alanine supplementation (e.g., 3–6 g/day for 2–4 weeks) has been reported to increase muscle carnosine levels, buffer acidity, and enhance exercise performance [6, 21‐24]. Research has indicated that length of time of supplementation affects the impact that beta-alanine supplementation has on muscle carnosine levels and potential ergogenic benefit [27, 60‐62]. Thus, it is possible that consuming 3 g/day of beta-alanine for 8-weeks could have provided ergogenic benefit during resistance-training. The PWS supplements also contained 2 g/day of creatine nitrate at approximately a 2:1 ratio of creatine to nitrate. While there is little evidence that ingesting 2 g/day of creatine for 8-weeks can markedly increase muscle creatine stores and/or affect exercise capacity, it is enough creatine to maintain creatine stores during training [7, 28]. The greater potential of ergogenic value from ingesting 2 g/day of creatine nitrate would theoretically be due to the nitrate content [46, 63]. There are a number of studies that indicate that nitrate supplementation prior to exercise (e.g., 300 mg) can improve exercise capacity [9, 11, 17‐20]. The amount of nitrate contained in the creatine nitrate supplement used (i.e., ~1 g/day) is more consistent with dietary recommendations to help control blood pressure [64, 65]. Nevertheless, there was sufficient amount of nitrates in the PWS to provide some ergogenic benefit particularly to endurance exercise performance. The PWS’s also contained 2 g/day of arginine alpha-ketoglutarate. Prior research has reported that dietary supplementation of higher doses of arginine alpha keto-glutarate (e.g., 12 g/day) can affect exercise capacity [12] although other studies with lower amounts have shown no benefit [66, 67]. Given that the PWS’s only contained 2 g/day of arginine alpha-ketoglutarate, it is likely that this nutrient provided minimal effects. Finally, one of the PWS’s contained 20 mg of synephrine (as Citrus Aurantium). Synephrine has been purported to suppress appetite [29] and promote weight loss through an enhanced thermogenesis [34‐36] with no negative effects on the cardiovascular system [37, 38, 68]. Theoretically, adding synephrine to the PWS would promote greater fat loss during training and a more optimal body composition.

Results of the present study provide some support that ingesting a PWS containing these nutrients enhanced training adaptations. Analysis of mean changes from baseline and 95% CI data (see Fig. 4) indicated that there was some evidence that 1RM strength gains were greater with PWS supplementation compared to placebo at both 4 and 8-weeks of training. There was also evidence that peak power, mean power, and total work during the anaerobic capacity sprint test was increased to a greater degree after 4-weeks of PWS + S supplementation (see Fig. 4). However, consistent with findings from Outlaw and colleagues [58], we did not observe a significant improvement in body composition changes in response to training as has previously been reported [56, 69]. This includes when adding 20 mg of synephrine (as Citrus Aurantium) to the formulation. These latter findings contrast reports suggesting that that Citrus Aurantium supplementation can promote weight and/or fat loss [36, 70] although it should be noted that the amount of synephrine provided (i.e., 20 mg/day) was less than that used in Kaats et al. study (i.e., 49 or 98 mg/day). Greenway and coworkers [71] reported that ingesting an herbal supplement containing 200 mg of green tea, 198 mg of caffeine (derived from guarana extract), and 9 mg of synephrine (derived from Citrus Aurantium) did not promote weight loss. Thus, it is possible that a higher dose of synephrine is needed to promote weight loss in individuals involved in intense training and/or addition of synephrine to a PWS has less effect in resistance-trained individuals.

One of the criticisms regarding PWS’s is that while there may be some ergogenic benefit, the short and/or long-term safety of taking these supplements is unknown [1]. Results of the present investigation indicate that the PWS’s studied were well-tolerated and did not adversely affect markers of health. In this regard, we observed no significant changes in resting hemodynamics, clinical blood markers, or a disproportional number of reported side effects in the PWS groups. These findings are consistent with a growing body of evidence that PWS supplementation appears to be safe and well-tolerated in apparently healthy individuals [56‐59, 69, 70, 72‐78] and that combination of these types of nutrients in PWS does not pose undue risk.