To examine whether common MRS techniques at 9.4 T yield a significant increase of amide signals, i.e. reduced amide-water proton exchange, in response to hypercapnia and hypothermia.Eleven mice were studied under isoflurane anaesthesia. After adjusting the rectal temperature at 37±1ºC, 27±1ºC, or 22±1ºC, the respective chemical shifts of the NAA amide signal at 7.83-7.84, 7.91-7.92, and 7.95-7.96 ppm confirmed the brain temperature to be within the target range. MRS (STEAM, TR/TE/TM = 6000/10/10 ms) with CHESS (90°-90°-180°, 350 Hz bandwidth) was performed. A (40 mm)³/(20 mm)³ VOI was centered on the forebrain/striatum, respectively. Metabolite concentrations were quantified by LCModel. Amide signal intensities of different compounds were normalized to those of NAA. Intracellular pH was estimated from the creatine phosphokinase equilibrium [H⁺] = ([ATP]×[Cr]×K´) / ([ADP]×[PCr]). [ATP]/[ADP] is calculated to 11.47 with K´ = 7.09×10^-9 at 37°C. For S_sat/S₀ and MTR_asym measurements [1], an off-resonance pulse (12 ms, 180°) was incorporated into 3D-FLASH (TR/TE=24/4.5 ms, α 5°, 250×250×500 µm³).Addition of 15% CO₂ to the inspiratory gas significantly lowered lactate and glutamate (Fig. 1a) and increased the amide signal intensity of glutamine H_Z [2,3] (relative to NAA amide) and of unspecific origin [1,4] (-NH_X). Their intensities are strongly correlated with each other (Fig. 1b). At 22°C, the signals of urea, H_Z, NAA amide, and -NH_X increased and a further amide signal of glutamine (H_E: 7.6 ppm) was identified. CO₂ increased the intensities of H_Z, H_E, and -NH_X, whereas interruption reduced these values (Fig. 1c). The intensities of H_Z and -NH_X as well as of H_E and -NH_X are strongly correlated with each other (Fig. 1d). CO₂ increased creatine and decreased phosphocreatine (Fig. 2a, b). pH decreased from 7.13±0.06 to 6.75±0.10. Interruption of CO₂ decreased creatine and increased phosphocreatine, while pH increased to 7.06±0.06. Glucose, lactate and glutamate concentrations changed significantly (Fig. 2c). The intensities of H_Z, H_E, and -NH_X were significantly correlated with pH (Figs. 3a, b). Figure 4a shows that CO₂ slightly altered S_sat/S₀. MTR_asym (Fig. 4b) decreased by 0.014±0.007 (2-5 ppm). Figures 4c,d show that a 10ºC reduction lowered the off-resonance saturation. The MTR decreased between 1-5 ppm by 0.032±0.02 and between -1 and -5 ppm by 0.030±0.01. MTR_asym (Fig. 4e) shows a slight drop. In other words, the increase of MTR with temperature can be ascribed to increased proton exchange and smaller proton relaxation rates R₁. Figures 4a,b,c,e also show that S_sat/S₀ is constantly higher in the aromatic range, i.e., MTR_asym is negative. This indicates that non-water protons in the brain in vivo are largely those of aliphatic compounds between -3.9 and -0.1 ppm, e.g., methyl (CH₃-) and methylene bridge (-CH₂-) protons. Accordingly, irradiation at -3.5 ppm saturates the water signal of the myelinated structures, rich in lipids and proteolipids, more than at +3.5 ppm (Fig. 5). MTR in the corpus callosum is 0.44±0.01 for -3.5 ppm and 0.26±0.04 for +3.5 ppm. The -3.5 ppm irradiation results in a significant white/gray matter contrast yielding MTR = 0.27±0.05 in the cerebral cortex, whereas the +3.5 ppm irradiation of aromatic/amide protons provides only a weak difference in the cerebral cortex with MTR =0.24±0.05. This observation is in agreement with the fact that neither the nucleic acid nor the protein content is significantly different between white and gray matter.This study confirms earlier observations of 8.3 ppm signals [4] and CEST-MRI [1] and further demonstrates that the 8.3 ppm signal intensities strongly correlate with both glutamine amide signals as well as with pH. The amide-water proton exchange in brain in vivo is best observable by MRS because (1) a number of short-T₂ protons around 6.8-8.3 ppm do not interfere with these resonances and (2) water protons can much more effectively be saturated than amide protons. This work also shows (3) the influence of temperature on the in vivo brain MRS signals of exchangeable protons as well as on S_sat/S₀ and MTR_asym and (4) that irradiation at -3.5 ppm (H₂O) provides a much better white/gray matter contrast than at +3.5 ppm (H₂O), which may be exploited for MRI studies of myelinated tissue. Given that CEST-MRI finds increasing applications and that the amide signal changes can be observed by commonly available MRS sequences, it is foreseeable that MRS of amide-water proton exchange as well as CEST-MRI will lead to new research applications of preclinical and clinical interest.This work identifies the effects of reduced pH and temperature on saturation transfer in MRS/MRI of mouse brain in vivo. Significant increases of amide signals, i.e. reduced amide-water proton exchanges, were observed.