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Background: Pre-workout supplements (PWS) have become increasingly popular with recreational and competitive athletes. While many ingredients used in PWS have had their safety assessed, the interactions when combined are less understood. Objective: The purpose of this study was to examine the safety of 1 and 2 servings of a PWS. Design: Forty-four males and females (24.4±4.6 years; 174.7±9.3 cm; 78.9±18.6 kg) from two laboratories participated in this study. Subjects were randomly assigned to consume either one serving (G1; n=14) or two servings (G2; n=18) of PWS or serve as an unsupplemented control (CRL; n=12). Blood draws for safety panels were conducted by a trained phlebotomist before and after the supplementation period. Results: Pooled data from both laboratories revealed significant group×time interactions (p<0.05) for mean corpuscular hemoglobin (MCH; CRL: 30.9±0.8-31.0±0.9 pg; G1: 30.7±1.1-30.2±0.7 pg; G2: 30.9±1.2-30.9±1.1 pg), MCH concentration (CRL: 34.0±0.9-34.4±0.7 g/dL; G1: 34.1±0.9-33.8±0.6 g/dL; G2: 34.0±1.0-33.8±0.8 g/dL), platelets (CRL: 261.9±45.7-255.2±41.2×103/µL; G1: 223.8±47.7- 238.7±49.6×103/µL; G2: 239.1±28.3-230.8±34.5×103/µL), serum glucose (CRL: 84.1±5.2-83.3±5.8 mg/dL; G1: 86.5±7.9-89.7±5.6 mg/dL; G2: 87.4±7.2-89.9±6.6 mg/dL), sodium (CRL: 137.0±2.7- 136.4±2.4 mmol/L; 139.6±1.4-140.0±2.2 mmol/L; G2: 139.0±2.2-138.7±1.7 mmol/L), albumin (CRL: 4.4±0.15-4.4±0.22 g/dL; G1: 4.5±0.19-4.5±0.13 g/dL; G2: 4.6±0.28-4.3±0.13 g/dL), and albumin:globulin (CRL: 1.8±0.30-1.8±0.28; G1: 1.9±0.30-2.0±0.31; G2: 1.8±0.34-1.8±0.34). Each of these variables remained within the clinical reference ranges. Conclusions: The PWS appears to be safe for heart, liver, and kidney function in both one-serving and two-serving doses when consumed daily for 28 days. Despite the changes observed for select variables, no variable reached clinical significance

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