Introduction: Creatine is a well-recognized, effective ergogenic agent that leads to an increase incellular creatine reserves. In recent years, guanidinoacetic acid (GAA) supplementation has beenpresented as a superior treatment for tackling cellular bioenergetic compared to creatine, and analternative solution that could eliminate potential shortcomings of creatine. The aim of this studyis to determine the effects of combined intake of creatine and GAA on anthropometric status,maximal, explosive and repetitive strength, aerobic endurance, as well as biochemical andindicators of tissue bioenergetics in healthy, well-trained men.Methods: 14 men between ages 18 and 30 (height 176.49±7.25 cm; body mass 79.86±13.86 kg)engaged in different physical activities for 5 or more hours per week participated in this research.In this randomized, double-blind, creatine-controlled cross-over study, the experimental groupingested a combination of creatine and GAA (3 g Cr + 1g GAA) for 4 weeks. The control groupconsumed an equimolar dose of creatine (4 g Cr) per day. Upon the end of the first phase andwash-out period (4 weeks), the groups switched treatments to ensure between-subjectscomparison. The daily dose included the intake of the powder dissolved in 250 ml of lukewarmwater immediately after night fasting. Anthropometric measurements, biochemical analyses,functional ability tests, and magnetic spectroscopy of muscle and brain tissue were conductedinitially, as well as after the completion of each research phase.Results: Creatine treatment led to a statistically significant increase in body weight compared tothe initial measurement (79,86±13,86 kg vs 81,48±13,22 kg; p=0,01), while these differences werenot determined after Cr+GAA treatment (80,6±13,68 kg; p>0,05). A significant increase in Crserum levels was observed in both groups (initial 20,87±5,3 μM/L vs Cr 44,45±16,53 μM/L;p=0.001, and Cr+GAA 39,83±12,37 μM/L; p=0,001), and no significant differences were observedbetween treatments (p>0,05). The increase in Crn serum levels was not statistically confirmed aftertreatments (p>0,05). Crn values were lower before supplementation compared to the Cr+GAAgroup (84±89 μM/L vs 88,45±6,44 μM/L), while creatine intake led to an additional increase(90,51±12,56 μM/L). Additionally, GAA and tHcy serum values did not differ significantly afterboth nutritional treatments compared to the initial measurement (p>0,05). Supplementation led toa statistically significant increase in strength. In the bench press, the values increased after creatineintake (97,08±23,67 kg vs 101,67±22,7 kg; p=0,003), and additionally after the Cr+GAAcombination (102,92±25,71 kg; p=0,004). In the front squat, a statistically significant increase instrength was observed after both treatments (initial 107,5±15,3 kg vs Cr 122,5±10,55 kg; p<0,00and Cr+GAA 121,25±8,29 kg; p<0,001), while the differences between treatments were notstatistically confirmed (p>0,05). In the vertical jump, a statistically significant decrease in jumpheight was observed after creatine treatment (57,06±6,02 cm vs 53,88±5,17 cm; p>0,001), andCr+GAA treatment (54,86±4,88 cm; p=0,006) compared to initial values. Furthermore, thecombination of Cr and GAA led to a statistically significant increase in gray matter creatine levels(Z=-2,02; p=0,043), but not creatine treatment alone (p>0,05) compared to initial values. Cr+GAAled to an increase in creatine levels by an additional 4,0% compared to creatine (initial 8,94±0,77mM vs Cr 9,09±0,93 mM; Cr+GAA 9,45±0,7 mM). The increase in white matter creatine levelswas not statistically confirmed after the supplementation intake (p>0,05), however, the creatineintake led to an increase of 7,0% compared to the initial measurement (7,60±0,63 mM vs 8,22±0,52mM), and the Cr+GAA treatment led to an additional increase of 6,3% compared to creatine alone(8,74±1,13 mM). The Cr+GAA combination achieved an additional 13,1% increase in creatinelevels in m. vastus medialis compared to creatine intake alone (initial 35,53±5,86 mM vs Cr36,03±4,91 mM; Cr+GAA 40,74±3,45 mM).Conclusion: The combination of creatine and GAA in a 3:1 ratio proved to be a superior treatmentfor improving the cellular creatine reserves in muscle and brain tissue compared to creatine alone.A new creatine formulation produced significant increases in upper and lower body strength,accompanied by significantly less weight gain compared to creatine after 4-weeks treatment inhealthy, physically active men. The investigated treatment significantly reduced the risk ofhyperhomocysteinemia compared to GAA intake alone.