In vivo ¹³C MRS provides unique insight into carbohydrate metabolism, such as the detection of natural abundance glycogen C₁ resonance in human muscle [1]. Nonetheless, the un-localized ¹³C spectra are dominated by intense ¹³C signals from subcutaneous adipose tissue [2], introducing baseline distortion and rendering difficult the detection of small ¹³C signals, such as the glycogen C₂-C₆ resonances due to their overlap with the glycerol C₂ and C₁,C₃ resonances [3]. Localized ¹³C MRS allows to remove the unwanted signal while the use of high magnetic field strength benefits from an improved signal to noise ratio (SNR) and spectral resolution, leading to an increased sensitivity and separation of carbon resonances [4]. Therefore, the aim of this study was to design a pulse-acquire sequence for localized ¹³C MRS using OVS and ISIS-1D schemes based on earlier reports [5, 6], and test the efficiency of distinct detection of glycogen C₂-C₆ resonances in vitro and in vivo in human muscle at 7T within SAR limitation.All experiments were performed on a 7T human MR scanner (Siemens Erlangen / Germany) with a ¹³C-linear / ¹H-quadrature RF surface coil (6cm diameter ¹³C-loop combined with 9cm diameter ¹H-loops). A small sphere (7mm diameter) containing 99% ¹³C-enriched formic acid was placed in the centre of the ¹³C-linear coil as an external reference. A pulse-acquire sequence for localized ¹³C MRS was designed with OVS and ISIS-1D [5, 6] schemes (Figure 1). In order to reduce the chemical shift displacement error, localization was applied using hyperbolic secant (HS) pulses of 5ms duration resulting in a 7kHz bandwidth (Figure 2), larger than the chemical shift range of the glycogen C₂-C₆ resonances (1.3 kHz). Localization was followed by symmetric adiabatic ¹³C excitation using two adiabatic half passage (AHP) pulses (2ms, 650Hz symmetric bandwidth) with inverted phases in alternate scans [7]. The WALTZ16 scheme [8] was applied during ¹³C signal acquisition for broadband ¹H-decoupling (90° pulse duration=0.9ms, resulting in a decoupling bandwidth of 600Hz), while the NOE scheme was applied prior to localization to achieve ¹³C signal enhancement (10 NOE pulses (pulse duration/delay=1/100ms)). The localization performance of the sequence was validated in vitro on a two-compartment phantom containing 800mM natural abundance glycogen (inner-compartment) and sunflower oil (outer-compartment). Three separate in vitro experiments were performed: 1) without localization, 2) with ISIS-1D localization in a slice parallel to the ¹³C coil plane, and 3) with ISIS-1D and three OVS double-bands ((VOI=70x30x30mm³), TR=1.19ms, 1000 averages, vector size=2048, BW=20kHz, decoupling duration=21ms, acquisition time=102ms). To validate the performance of the sequence, localized ¹³C MR spectra were acquired in vivo on the human calf of two healthy male volunteers who gave informed consent according to the procedure approved by the local ethics committee. Localization was applied in vivo using ISIS-1D (coronal slice parallel to the ¹³C coil plane) and two OVS double-bands ((2 transversal and 2 sagital), VOI=80x60x40mm³, TR=1s, 3000 averages, vector size=2048, BW=20kHz, decoupling duration=21ms, acquisition time=102ms). All in vitro and in vivo ¹³C spectra were acquired using the glycerol C₂ resonance at the centre of the spectrum. First and second order shim were adjusted using FAST(EST)MAP [9] with VOI=80x60x40mm³.Shimming resulted in a water line-width of 25 Hz. Un-localized ¹³C MRS in vitro (Figure 3-a) revealed peak resonances in agreement with the literature [2, 3]. Localized ¹³C MRS in vitro (Figure 3-c) demonstrated efficient suppression of glycerol C₂ (69.5ppm) and C₁,C₃ (61.4ppm) resonances using both OVS and ISIS-1D schemes, allowing simultaneous detection of well resolved carbons of glycogen C₄ (78ppm) and C₆ (61ppm), and clustered carbons of glycogen C₃ (74ppm) and C₂,C₅ (72.2ppm), in contrast to that using only ISIS-1D scheme (Figure 3-b) in which localization was incomplete. Furthermore, the baseline appeared to be improved when using localization. Localized ¹³C MRS in vivo revealed well resolved glycogen C₂-C₆ resonances (Figure 4), while some glycerol C₂ and C₁,C₃ remained in the muscle, ascribed to the presence of intra- and extra-myocellular lipids in muscle and interstitial adipose tissue, respectively [6]. In addition, the localization sequence efficiently suppressed the intense lipid resonance at 130 ppm, as no residual lipid signal was observed on the ¹³C localized in vivo spectrum (Figure 5).We conclude that localized ¹³C MRS is feasible at 7T using an optimized pulse-sequence including both OVS and ISIS-1D schemes for ¹³C localization followed by symmetric adiabatic ¹³C excitation, resulting in efficient suppression of glycerol C₂ and C₁,C₃ resonances, as well as intense lipid resonance at 130 ppm, and this will allow further extension of this technique for ¹³C MRS measurements such as in human brain.