A study was conducted to evaluate the effects of lowering dietary fishmeal (FM) and crude protein (CP) levels, while maintaining essential amino acid levels, on growth performance, body composition, muscle metabolic gene expression, and chronic stress response of rainbow trout, Oncorhynchus mykiss, with and without handling stress. Eight experimental diets (isocaloric) with a 4 × 2 factorial design were formulated to contain two levels of FM (20%, 5%) and four levels of CP (48%, 45%, 42%, 39%). Diets were supplemented with increasing levels of amino acids to maintain dietary essential amino acid (EAA) levels. Trout (34.8 ± 0.3 g) were fed to apparent satiation twice daily for nine weeks to assess growth performance under laboratory rearing conditions, and then for an additional six weeks with and without exposure to handling stress (30 s of chasing followed by 30 s of netted air exposure) twice per week. The 9-week growth trial demonstrated that reducing dietary FM levels from 20% to 5% significantly reduced fish growth and increased feed conversion ratio (P < .05). Reducing dietary CP levels from 48% to 42% did not affect trout growth. A dietary FM level of 20% significantly increased whole-body dry matter, CP and total EAAs (P < .05) compared to 5% FM inclusion while increasing dietary CP level significantly decreased dry matter, crude fat, and gross energy but increased total EAAs (P < .05). Reducing FM and CP levels had no effect on measured stress indices of plasma cortisol, glucose and lysozyme activity (P > .05) after 6-weeks of repeated handling stress. The expression of genes in the gcn2/eif2α/atf4 pathway, triggered in response to protein or amino acid starvation, were evaluated. General control nonderepressible 2 (gcn2) decreased with increasing dietary CP level above 42% (P < .05), but there were no dietary effects (FM or CP levels) on eif2α (eukaryotic initiation factor 2α) or atf4 (activating transcription factor 4) expression. In total, gene expression results suggest amino acid limitations on muscle protein metabolism as a result of feeding diets below 42% CP, even when supplemented with synthetic EAA to meeting published dietary requirements. In conclusion, our study demonstrated that 5% dietary FM is insufficient for maximal growth performance, while diets balanced for EAAs show an opportunity to reduce CP level from 48% to 42% without any reduction in growth performance, body composition, metabolic amino acid sufficiency or tolerance to chronic stress. Below 42% CP, reduced growth indices suggest an imbalance in EAA availability.