diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16-bun/proxy.ts b/dev-packages/e2e-tests/test-applications/nextjs-16-bun/proxy.ts
index 60722f329fa0..e2d263c4d80c 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16-bun/proxy.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16-bun/proxy.ts
@@ -7,6 +7,15 @@ export async function proxy(request: NextRequest) {
   Sentry.setTag('my-isolated-tag', true);
   Sentry.setTag('my-global-scope-isolated-tag', getDefaultIsolationScope().getScopeData().tags['my-isolated-tag']); // We set this tag to be able to assert that the previously set tag has not leaked into the global isolation scope
 
+  // Streamed spans carry no scope tags, so the tests read the isolation state from this attribute instead
+  const activeSpan = Sentry.getActiveSpan();
+  if (activeSpan) {
+    Sentry.getRootSpan(activeSpan).setAttribute(
+      'isolation_scope.is_default',
+      Sentry.getIsolationScope() === getDefaultIsolationScope(),
+    );
+  }
+
   if (request.headers.has('x-should-throw')) {
     throw new Error('Middleware Error');
   }
diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16-bun/tests/middleware.test.ts b/dev-packages/e2e-tests/test-applications/nextjs-16-bun/tests/middleware.test.ts
index 068c5454176e..2b56d36de0ce 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16-bun/tests/middleware.test.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16-bun/tests/middleware.test.ts
@@ -14,4 +14,7 @@ test('Should create a span for middleware', async ({ request }) => {
   expect(middlewareSpan.status).toBe('ok');
   expect(getSpanOp(middlewareSpan)).toBe('middleware');
   expect(middlewareSpan.attributes['sentry.segment.name.source']?.value).toBe('route');
+
+  // Assert that isolation scope works properly
+  expect(middlewareSpan.attributes['isolation_scope.is_default']).toEqual({ value: false, type: 'boolean' });
 });
diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/middleware.ts b/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/middleware.ts
index f5980e4231c1..a6d31cc8c66f 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/middleware.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/middleware.ts
@@ -7,6 +7,15 @@ export async function middleware(request: NextRequest) {
   Sentry.setTag('my-isolated-tag', true);
   Sentry.setTag('my-global-scope-isolated-tag', getDefaultIsolationScope().getScopeData().tags['my-isolated-tag']); // We set this tag to be able to assert that the previously set tag has not leaked into the global isolation scope
 
+  // Streamed spans carry no scope tags, so the tests read the isolation state from this attribute instead
+  const activeSpan = Sentry.getActiveSpan();
+  if (activeSpan) {
+    Sentry.getRootSpan(activeSpan).setAttribute(
+      'isolation_scope.is_default',
+      Sentry.getIsolationScope() === getDefaultIsolationScope(),
+    );
+  }
+
   if (request.headers.has('x-should-throw')) {
     throw new Error('Middleware Error');
   }
diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/tests/middleware.test.ts b/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/tests/middleware.test.ts
index c677115cd682..78b27c8427d9 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/tests/middleware.test.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16-cf-workers/tests/middleware.test.ts
@@ -30,6 +30,9 @@ test.skip('Should create a span for middleware', async ({ request }) => {
   expect(middlewareSpan.status).toBe('ok');
   expect(getSpanOp(middlewareSpan)).toBe('middleware');
   expect(middlewareSpan.attributes['sentry.segment.name.source']?.value).toBe('route');
+
+  // Assert that isolation scope works properly
+  // expect(middlewareSpan.attributes['isolation_scope.is_default']).toEqual({ value: false, type: 'boolean' });
 });
 
 // TODO: Middleware tests need SDK adjustments for Cloudflare Workers edge runtime
diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16/app/api/endpoint-behind-middleware/route.ts b/dev-packages/e2e-tests/test-applications/nextjs-16/app/api/endpoint-behind-middleware/route.ts
index 2733cc918f44..e2e27470d279 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16/app/api/endpoint-behind-middleware/route.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16/app/api/endpoint-behind-middleware/route.ts
@@ -1,3 +1,17 @@
+import { getDefaultIsolationScope } from '@sentry/core';
+import * as Sentry from '@sentry/nextjs';
+
 export function GET() {
+  // Streamed spans carry no scope tags, so the middleware test reads from these attributes whether the
+  // isolation scope marked in `proxy.ts` leaked into this request
+  const activeSpan = Sentry.getActiveSpan();
+  if (activeSpan) {
+    const isolationScope = Sentry.getIsolationScope();
+    Sentry.getRootSpan(activeSpan).setAttributes({
+      'isolation_scope.is_default': isolationScope === getDefaultIsolationScope(),
+      'isolation_scope.has_proxy_marker': 'proxy-marker' in isolationScope.getScopeData().contexts,
+    });
+  }
+
   return Response.json({ name: 'John Doe' });
 }
diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16/proxy.ts b/dev-packages/e2e-tests/test-applications/nextjs-16/proxy.ts
index 60722f329fa0..2daf05c0c50d 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16/proxy.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16/proxy.ts
@@ -7,6 +7,19 @@ export async function proxy(request: NextRequest) {
   Sentry.setTag('my-isolated-tag', true);
   Sentry.setTag('my-global-scope-isolated-tag', getDefaultIsolationScope().getScopeData().tags['my-isolated-tag']); // We set this tag to be able to assert that the previously set tag has not leaked into the global isolation scope
 
+  const isolationScope = Sentry.getIsolationScope();
+  // Marks this request's isolation scope so the route handler behind the proxy can check it did not leak there
+  isolationScope.setContext('proxy-marker', { set: true });
+
+  // Streamed spans carry no scope tags, so the tests read the isolation state from these attributes instead
+  const activeSpan = Sentry.getActiveSpan();
+  if (activeSpan) {
+    Sentry.getRootSpan(activeSpan).setAttributes({
+      'isolation_scope.is_default': isolationScope === getDefaultIsolationScope(),
+      'isolation_scope.has_proxy_marker': 'proxy-marker' in isolationScope.getScopeData().contexts,
+    });
+  }
+
   if (request.headers.has('x-should-throw')) {
     throw new Error('Middleware Error');
   }
diff --git a/dev-packages/e2e-tests/test-applications/nextjs-16/tests/middleware.test.ts b/dev-packages/e2e-tests/test-applications/nextjs-16/tests/middleware.test.ts
index 9a0214980632..765d3961238f 100644
--- a/dev-packages/e2e-tests/test-applications/nextjs-16/tests/middleware.test.ts
+++ b/dev-packages/e2e-tests/test-applications/nextjs-16/tests/middleware.test.ts
@@ -7,6 +7,10 @@ test('Should create a span for middleware', async ({ request }) => {
     spans.some(span => span.name === 'middleware GET' && span.is_segment),
   );
 
+  const routeSpanPromise = waitForStreamedSpan('nextjs-16', span => {
+    return span.name === 'GET /api/endpoint-behind-middleware' && span.is_segment;
+  });
+
   const response = await request.get('/api/endpoint-behind-middleware');
   expect(await response.json()).toStrictEqual({ name: 'John Doe' });
 
@@ -24,6 +28,17 @@ test('Should create a span for middleware', async ({ request }) => {
   // `wrapMiddlewareWithSentry` wrapper used to start a second, redundant one nested inside it.
   const nestedMiddlewareSpans = spans.filter(span => getSpanOp(span) === 'middleware' && !span.is_segment);
   expect(nestedMiddlewareSpans).toHaveLength(0);
+
+  // Assert that isolation scope works properly
+  expect(middlewareSpan.attributes['isolation_scope.is_default']).toEqual({ value: false, type: 'boolean' });
+  expect(middlewareSpan.attributes['isolation_scope.has_proxy_marker']).toEqual({ value: true, type: 'boolean' });
+
+  // Scope data set in middleware must not leak into other requests (e.g. via a shared scope when the middleware
+  // runs in a detached context - https://github.com/vercel/next.js/pull/95306). The route handler exposes it
+  // via the same attributes.
+  const routeSpan = await routeSpanPromise;
+  expect(routeSpan.attributes['isolation_scope.is_default']).toEqual({ value: false, type: 'boolean' });
+  expect(routeSpan.attributes['isolation_scope.has_proxy_marker']).toEqual({ value: false, type: 'boolean' });
 });
 
 test('Faulty middlewares', async ({ request }) => {