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The spline reconstruction technique (SRT) is a fast algorithm based on a novel numerical implementation of an analytic representation of the inverse Radon transform. The purpose of this study was to compare the SRT, filtered back-projection (FBP), and the Tera-Tomo 3D algorithm for various iteration numbers, using small-animal dynamic PET data obtained from a Mediso nanoScan^® PET/CT scanner. For this purpose, Patlak graphical kinetic analysis was employed to noninvasively quantify the myocardial metabolic rate of glucose (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mi>M</mi><mi>R</mi><mi>G</mi><mi>l</mi><mi>u</mi></mrow></semantics></math></inline-formula>) in seven male C57BL/6 mice (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mi>n</mi><mo>=</mo><mn>7</mn></mrow></semantics></math></inline-formula>). All analytic reconstructions were performed via software for tomographic image reconstruction. The analysis of all PET-reconstructed images was conducted with PMOD software (version 3.506, PMOD Technologies LLC, Fällanden, Switzerland) using the inferior vena cava as the image-derived input function. Statistical significance was determined by employing the one-way analysis of variance test. The results revealed that the differences between the values of <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mi>M</mi><mi>R</mi><mi>G</mi><mi>l</mi><mi>u</mi></mrow></semantics></math></inline-formula> obtained via SRT versus FBP, and the variants of he Tera-Tomo 3D algorithm were not statistically significant (<i>p</i> > 0.05). Overall, the SRT appears to perform similarly to the other algorithms investigated, providing a valid alternative analytic method for preclinical dynamic PET studies.