GABA+ (GABA+ macromolecules (MM)) – along with Glutamate + Glutamine (Glx) – are measured through spectral editing methods, such as the MEGA-SPECIAL (MSpc) sequence⁽¹⁾. Although studies have characterized short and long term reproducibility of GABA+^(2-3) and compared effects of using different analysis strategies, this has not been done for MM-suppressed GABA. Gender-related differences in GABA+ concentration have also been observed⁽³⁾. The relative contribution of MM, however, varies between 40-60%⁽¹⁾ of the total GABA+ signal in individuals and may account for some of these differences. Furthermore, instability in the initial prepared shim of the MRI scanner, which arises due to several factors including incidental motion, may alter concentration estimates⁽⁴⁾. The aims of this study were (i) to use MM-suppressed navigated MSpc⁽⁴⁾, which performs real time motion and shim correction, to compare intra-subject short term reproducibility of GABA and Glx concentrations obtained using GANNET⁽⁵⁾, LCModel⁽⁶⁾, and jMRUI⁽⁷⁾, and (ii) to investigate gender-related differences in GABA and Glx concentrations in healthy subjects. All protocols and experiments were approved by the local institutional Human Research Ethics Committee. Scans were performed on a Siemens Allegra 3T scanner. 26 healthy subjects (13 female; age range: 20-29 years) with no history of neurological or psychiatric disorders were scanned. A T1-weighted and 4 motion and shim corrected MSpc⁽⁴⁾ scans were acquired in a single session. Two spectra were acquired from each of two regions, the anterior cingulate cortex (ACC) and medial-parietal cortex (PAR), with the order interleaved. The MSpc parameters were as follows: TR/TE 4000/68 ms and 160 averages. The voxel sizes were: ACC: 20 × 30 ×40 mm³ and PAR: 30 × 30 ×30 mm³. Water unsuppressed data with four averages were acquired from every region. Water scaled metabolite concentrations were derived from LCModel, GANNET and AMARES in jMRUI^(5-7). The T1 images were segmented using SPM12 (http://www.fil.ion.ucl.ac.uk/spm/software) to determine grey matter (GM) white matter (WM) and CSF fractions. The concentrations were corrected for CSF contamination and relaxation effects⁽³⁾. Reproducibility was compared for GABA and Glx concentrations derived using each of the analysis packages by computing for each subject the coefficient of variation (CV = mean/standard deviation (SD)) from the pair of measurements in each region. The mean CV across subjects were compared for each analysis package. Mean GABA and Glx concentrations derived from LCModel in each region were compared in males and females using a 2-tailed t-test. Linear regression was used to control for differences in GM ratio (GM/(GM+WM)) between males and females. Figure 1 shows a typical MSpc edited spectrum with GABA at 3 ppm and Glx at 3.75 ppm, which were observed in all subjects. Table 1 shows how the CV differs in each region for GABA and Glx concentrations derived using each of the analysis packages. Reproducibility of GABA and Glx was highest in both regions when using LCModel, followed by JMRUI and GANNET. GM fraction tended to be higher in males (ACC; PAR: p=0.06; p=0.10), while WM fraction in females was higher in ACC (p=0.01) and tended to be higher in PAR (p=0.06). Males had significantly higher GABA in PAR (Figure 2), which remained significant after controlling for differences in GM ratio (β = -0.46, p = 0.031). No gender-related differences were found in Glx concentration. GABA acquisition without MM requires editing pulses to be applied consistently at 1.7 ppm⁽¹⁾, which can be hampered by motion and motion-related magnetic field inhomogeneity. In our study we implemented a motion and shim navigated MSpc sequence to allow prospective motion and frequency correction for effective MM suppression⁽⁴⁾. The resulting GABA and Glx peaks were highly reproducible using LCModel, similar to results from a previous study⁽³⁾. The higher GABA concentration found here in males in PAR is in agreement with findings from a previous study using MEGA-PRESS that reported higher GABA+ concentrations in males in a region in the left dorsolateral prefrontal cortex⁽³⁾. Another study using MM-suppressed MEGA-PRESS⁽⁸⁾ did not find gender differences in the ACC. These results suggest that gender differences may be localised. Further, the fact that gender-related differences have been observed in GABA+⁽³⁾ and GABA may indicate that contributions from MM are similar in males and females so that gender differences are not impacted.The shim and motion navigated MSpc sequence produced well edited GABA and Glx spectra. Both GABA and Glx can be quantified with best reproducibility using LCModel. Regional gender-related differences in GABA emphasize the importance of gender-matching for studies investigating differences in GABA concentrations.