Objective: Cerebral photobiomodulation (PBM) improves mood and cognition. Cerebral metabolic enhancement is a mechanism proposed to underlie PBM effects. No PBM studies to date have applied phosphorus magnetic resonance spectroscopy (P-31 MRS), which can be used to assess metabolic intermediates such as phosphocreatine (PCr) and adenosine triphosphate, the latter of which is elevated by PBM. Accordingly, we used 9.4 Tesla P-31 MRS to characterize effects of single and repeat cerebral PBM treatments on metabolism. PBM was delivered to healthy adult beagles in the form of transcranial laser treatment (TLT) at a wavelength of 808 nm, which passes safely through the skull and activates cytochrome C oxidase, a mitochondrial respiratory chain enzyme. Methods: Isoflurane-anesthetized subjects (n = 4) underwent a baseline P-31 MRS scan followed by TLT applied sequentially for 2 min each to anterior and posterior cranium midline locations, to irradiate the dorsal cortex. Subjects then underwent P-31 MRS scans for 2h to assess acute TLT effects. After 2 weeks of repeat TLT (3 times/week), subjects were scanned again with P-31 MRS to characterize effects of repeat TLT. Results: TLT did not induce acute P-31 MRS changes over the course of 2 h in either scan session. However, after repeat TLT, the baseline PCr/beta-nucleoside triphosphate ratio was higher than the scan 1 baseline (p < 0.0001), an effect attributable to increased PCr level (p < 0.0001). Conclusions: Our findings are consistent with reports that bioenergetic effects of PBM can take several hours to evolve. Thus, in vivo P-31 MRS may be useful for characterizing bioenergetic effects of PBM in brain and other tissues.