G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT₂GGTGTGGT₂GG) that is referred to as G2. The sequences in italics, TGT and T₂, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T₂ loops (T₄, U₂), or in both loops (UGU and U₂, UUU and U₂). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the “chair” conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (T_Ms) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with T_Ms in the range from 43.2 to 56.5 °C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K⁺ ions (0.1−0.6 mol K⁺/mol), and a variable uptake/release of water molecules (−6 to 8 mol H₂O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base−base stacking within the loops) and their hydration effects.